Xenorhabdus  sp. genome sequences and uses thereof

ABSTRACT

The present invention relates to a nucleic acid sequences from  Xenorhabdus  that encodes a GroEL protein. The invention encompasses nucleic acid molecules present in non-coding regions as well as nucleic acid molecules that encode proteins, fragments of proteins, tRNA&#39;s, fragments of tRNA&#39;s, rRNA&#39;s and fragments of rRNA&#39;s. In addition, protein and fragments of proteins so encoded and antibodies capable of binding the proteins are encompassed by the present invention. The invention also relates to methods of using the disclosed nucleic acid molecules, proteins, fragments of proteins, RNA&#39;S, and antibodies, for example, for gene identification and analysis, and preparation of constructs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part (CIP) application of U.S.Non-provisional Application Ser. No. 12/385,507, filed Apr. 9, 2009,which is a continuation of U.S. Non-provisional application Ser. No.12/289,606, filed Oct. 30, 2008, which is a continuation of U.S.Non-provisional application Ser. No. 09/897,516, filed Jun. 29, 2001,which claims priority of U.S. Provisional Application 60/215,161, filedJun. 30, 2000. Each of the aforementioned US applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to genomic nucleic acid sequences fromXenorhabdus sp., in particular, and to Photorhabdus sp., and includesnucleic acid molecules present in both coding and in non-coding regions.Nucleic acid sequences that encode proteins and/or enzymes andhomologues and fragments thereof are encompassed by the inventionincluding but not limited to insect inhibitory proteins, proteinscapable of conferring antibiotic resistance, microbial inhibitoryproteins including bactericidal, bacteriostatic, fungicidal, andfungistatic proteins, proteins capable of conferring resistance to heavymetals or other toxic compositions, proteins and compositions capable ofconferring pharmaceutical advantages such as antineoplastic, acaricidal,anti-inflammatory and anti-ulcerogenic properties, polyketide synthases,transposons and mobile genetic elements and their correspondingtransposases, excisases and integrases, phage and phage particleproteins, other useful Xenorhabdus, Photorhabdus, Serratia, Yersinia,Salmonella, E. coli, and Erwinia sp. protein homologues, ribosomal RNA(rRNA), and transfer RNA (tRNA). In addition, proteins and fragmentsthereof so encoded and antibodies capable of binding the proteins areencompassed by the present invention. The invention also relates tomethods of using the disclosed nucleic acid molecules, proteins,fragments of proteins, and antibodies, for example, for geneidentification and analysis, preparation of constructs, transformationof cells with nucleotide compositions disclosed herein to produceXenorhabdus proteins or fragments thereof, in particular novel insectinhibitory, bactericidal, fungicidal and nematicidal proteins.

BACKGROUND OF THE INVENTION

Xenorhabdus sp. and Photorhabdus sp. strains have previously been shownto produce an array of extracellular proteins and small molecules orsecondary metabolites having specialized functions. Among the morecommercially interesting are proteins and small molecules havingantibiotic properties or proteins which exhibit insect inhibitoryactivity. A small number of insect inhibitory proteins have previouslybeen identified from these bacteria, symbionts of insect-parasiticnematodes. In view of the biotechnology methods which are now available,such proteins and compositions have great potential for use asbiologically safe and effective pest control agents. Unlike chemicalpesticide compositions, these proteins have no effect upon theenvironment in general, can be targeted to direct their effect primarilyupon target insect species, and have no effect on non-target species.These proteins are comparable in nature to Bacillus thuringiensis (BT)proteins, which are the most widely used biological insect pest controlagents derived from various strains of Bacillus thuringiensis. BTcompositions have been in commercial use for more than twenty years astopically applied insect control agents and more recently genes encodingvarious BT proteins have been expressed in transgenic plants, and inparticular in agronomically important crops such as soybean, corn,wheat, rice, and cotton. However, one issue related to the use of BTproteins is resistance management. The concern is that target insectpests feeding on a plant expressing a single BT protein that isgenerally effective against that pest species will develop resistance tothe protein in some calculable period of time. The answer to thisproblem has been to include in the plant another BT protein also toxicto the same target pest species. The idea is similar in nature tobacterial resistance management, in that the development of resistanceto either of the BT proteins will be delayed because pest will notproduce progeny that are resistant to either of the BT proteins, inparticular if the two proteins that are expressed in the plant havedifferent modes of action or bind different receptors in the insectmidgut. Unfortunately, BT proteins are highly related and often it isdifficult to distinguish whether two BT proteins toxic to the sameinsect species have different modes of action. Thus, even though a greatvariety of BT proteins have been identified, characterized andcategorized into distinct classes of proteins, all appear to act in avery similar fashion. Therefore, a different resistance managementstrategy which takes advantage of insect inhibitory proteins derivedfrom distinct microbial sources other than Bacillus thuringiensis wouldbe desirable. Insect inhibitory proteins isolated from Xenorhabdus andPhotorhabdus species of bacteria seem to have all the prerequisites forthe delivery of novel genes for transgenic expression of insect pestinhibiting proteins to provide pest resistance to plants, either aloneor in combination with Bacillus thuringiensis insecticidal crystalproteins.

Xenorhabdus sp. is a Gram-negative bacterium, member of the family ofEnterobacteriaceae, and symbiotically associated with nematodes of thegenus Steinernema. The nematode-bacterial complex can be characterizedas an obligate and lethal parasitic relationship, specializing inparasitizing and proliferating in soil insect larvae. Infective,non-feeding stages of these nematodes live in soil and carry theirnematode-genus-specific symbiotic bacteria in the gut. It is believedthat the nematodes actively search for the appropriate insect host,invade the insect larvae through natural openings or lesions in thecuticle and, once inside the hemolymphe, release their symbioticbacteria. The nematode-bacterial complex secretes a variety of highlyefficient extracellular metabolites and proteins exhibiting insecticinhibitory, bactericidal, fungicidal and nematicidal properties tosecure the larval mass as a source of nutrition. An array ofextracellular enzymes such as lipases, phospholipases, proteases,nucleases as well as several broad spectrum antibiotics, and antifungaland nematicidal compositions are also secreted (Boemare & Akhurst, J.Gen. Microbiol. 134: 751-761 (1988); Li et al., Can. J. Microbiol.43(8):770-773 (1997); McInerney et al., J. Nat. Prod. 54(3):774-84(1991); McInerney et al., J. Nat. Prod. 54(3):785-95 (1991); Sundar andChang, J. Gen. Microbiol. 139 (Pt 12):3139-48 (1993)). It has beendiscovered that some compounds secreted by Xenorhabdus exhibitanti-neoplastic (U.S. Pat. No. 5,827,872), acaricidal, anti-inflammatoryand anti-ulcerogenic properties (U.S. Pat. No. 4,837,222). U.S. Pat. No.6,048,838 describes insect inhibitory proteins which exhibit a molecularweight of greater than 100 kDa produced by Xenorhabdus sp. which areorally active against a variety of insect species including the ordersLepidoptera, Coleoptera, Diptera, and Acarina.

The nomenclature and taxonomic characterization of Xenorhabdus hasrecently been subject to innovations in the state of the art. The genusPhotorhabdus was separated from the genus Xenorhabdus in 1993 because ofsignificant differences in biochemical and molecular characterization(Boemare et al., Int. J. Syst. Bacteriol. 43: 249-255 (1993)).Xenorhabdus exists of at least 4 known species: X. nematophilus, X.beddingii, X. poinarii and X. bovienii. (Brunel et al., Appl. &Environm. Microbiol. 63: 574-580 (1997)). Species of Xenorhabdus as wellas Photorhabdus species can be distinguished from each other byrestriction analysis of thermally amplified 16S rRNA genes (Brunel etal., Appl. Environm. Microbiol. 63: 574-580 (1997)).

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteriaassociated with entomopathogenic nematodes appears to be quite large.The genus Xenorhabdus appears more diverse than the genus Photorhabdus,and for both genera, the bacterial genotype diversity is in congruencewith the host-nematode taxonomy. It has been found that the occurrenceof symbiotic bacterial genotypes was related to the ecologicaldistribution of host nematodes (Fisher-Le Saux et al., Appl. Environ.Microbiol. 64(11):4246-54 (1998)). Xenorhabdus bacteria isolated fromthe same geographical location seem to be more similar to each other,regardless of nematode species than bacteria from one nematode speciesfound in very diverse geographical locations (Liu et al., Intl. J. Syst.Bacteriol. 47:948-951; 1997).

Therefore, there is a great deal of interest in identifying the genesthat encode new insect inhibiting proteins, and proteins involved in thebiosynthetic pathways of novel antibiotics produced by Xenorhabdus andPhotorhabdus bacteria, as well as other useful proteins. Sequencing ofthe entire genome of Xenorhabdus would facilitate such an endeavor,because it would allow dissection and analysis of the genome intodiscrete genes encoding proteins having beneficial properties asdescribed herein.

SUMMARY OF THE INVENTION

The present invention provides an isolated and purified nucleic acidmolecule having a nucleotide sequence, wherein: (1) the nucleotidesequence hybridizes under stringent conditions to a second isolated andpurified nucleic acid molecule comprising SEQ ID NO:1 or the complementthereof; (2) the nucleotide sequence is a portion of SEQ ID NO:1; or (3)the nucleotide sequence is the complement of (1) or (2).

The present invention also provides an isolated and purified nucleicacid molecule comprising a nucleotide sequence, wherein: (1) thenucleotide sequence hybridizes under stringent conditions to a secondisolated and purified nucleic acid molecule, wherein the hybridizingportion of the nucleotide sequence of the second nucleic acid moleculeencodes a polypeptide or protein having an amino acid sequence of SEQ IDNO:2; (2) the nucleotide sequence encodes a polypeptide or protein,wherein the amino acid sequence of the polypeptide or protein issubstantially identical to SEQ ID NO:2; or (3) the nucleotide sequenceis the complement of (1) or (2). In alternative embodiments, the aminoacid sequence of the above described polypeptide or protein is at least70% identical, at least 80% identical, at least 85% identical, at least90% identical, or at least 95% identical to an amino acid sequence ofSEQ ID NO:2. In a preferred embodiment, the amino acid sequence of theabove described polypeptide or protein is SEQ ID NO:2 or SEQ ID NO:2with conservative amino acid substitutions.

The present invention further provides a method for obtaining a nucleicacid molecule comprising a nucleotide sequence encoding a polypeptide orprotein having an amino acid sequence that is at least 70% identical toSEQ ID NO:2.

The present invention, in another aspect, provides a substantiallypurified polypeptide or protein comprising an amino acid sequence,wherein the amino acid sequence is defined as follows: (1) the aminoacid sequence is encoded by a first nucleotide sequence whichspecifically hybridizes to the complement of a second nucleotidesequence of SEQ ID NO:1; (2) the amino acid sequence is encoded by athird nucleotide sequence that is at least 50% identical to SEQ ID NO:1;or (3) the amino acid sequence is at least 70% identical to SEQ ID NO:2.In alternative embodiments, the above described third nucleotidesequence is at least 55% identical, at least 60% identical, at least 65%identical, at least 70% identical, at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, or at least95% identical to SEQ ID NO:1; and, the above described third nucleotidesequence is SEQ ID NO:1. In a preferred embodiment, the above describedamino acid sequence is at least 70% identical, at least 80% identical,at least 90% identical, or at least 95% identical to SEQ ID NO:2.

The present invention also provides a recombinant construct comprising:(A) a promoter region which functions in a host cell to cause theproduction of a mRNA molecule; which is operably linked to (B) astructural nucleotide sequence, wherein the structural nucleotidesequence is substantially identical to SEQ ID NO:1; which is operablylinked to (C) a 3′ non-translated sequence that functions in said cellto cause termination of transcription.

The present invention also provides a recombinant construct comprising:(A) a promoter region which functions in a host cell to cause theproduction of a mRNA molecule; which is operably linked to (B) astructural nucleotide sequence, wherein the structural nucleotidesequence encodes a polypeptide or protein having an amino acid sequencesubstantially identical to SEQ ID NO:2; which is operably linked to (C)a 3′ non-translated sequence that functions in said cell to causetermination of transcription.

The present invention also provides a recombinant construct comprising:(A) a promoter region which functions in a host cell to cause theproduction of a mRNA molecule, wherein the promoter region is locatedwithin SEQ ID NO:1 or complements thereof; which is linked to (B) astructural nucleotide sequence encoding a polypeptide; which is linkedto (C) a 3′ non-translated sequence that functions in said cell to causetermination of transcription.

The present invention also provides a transformed cell having anexogenous nucleic acid molecule which comprises: (A) a promoter regionwhich functions in said cell to cause the production of a mRNA molecule;which is operably linked to (B) a structural nucleic acid molecule,wherein the structural nucleotide is substantially identical to SEQ IDNO:1; which is operably linked to (C) a 3′ sequence that functions insaid cell to cause termination of transcription.

The present invention also provides a transformed cell having anexogenous nucleic acid molecule which comprises: (A) a promoter regionwhich functions in said cell to cause the production of a mRNA molecule;which is operably linked to (B) a structural nucleic acid molecule,wherein the structural nucleotide encodes a polypeptide or proteinhaving an amino acid sequence substantially identical to SEQ ID NO:2;which is operably linked to (C) a 3′ sequence that functions in saidcell to cause termination of transcription.

The present invention also provides a transformed cell having anexogenous nucleic acid molecule which comprises: (A) a promoter regionwhich functions in said cell to cause the production of a mRNA molecule,wherein the promoter region is located within SEQ ID NO:1 or complementsthereof; which is operably linked to (B) a structural nucleotidesequence encoding a polypeptide; which is operably linked to (C) a 3′sequence that functions in said cell to cause termination oftranscription.

The present invention also provides a plant cell, a mammalian cell, abacterial cell, an algal cell, an insect cell and a fungal celltransformed with an isolated nucleic acid molecule of the presentinvention.

The invention also provides isolated nucleic acid molecules comprisingnucleotide sequences encoding polypeptides or proteins exhibiting insectinhibitory activity, wherein said activity is manifested by inhibitingthe growth or development of, or contributing substantially to, orcausing the death of a Coleopteran, a Dipteran, a Lepidopteran, aHemipteran, a Hymenopteran, or a sucking and piercing insect or insectlarvae thereof. Also provided are nucleotide sequences encoding novelproteins comprising polypeptides which augment the activity ofpolypeptides exhibiting insect inhibitory activity when fed toColeopteran, Dipteran, Lepidopteran, Hemipteran, Hymenopteran, orsucking and piercing insects or insect larvae thereof.

The present invention also provides a method for using insect inhibitoryproteins for controlling target insect pests, i.e. also known as insectpest control.

The present invention also provides a computer readable medium havingrecorded thereon one or more of the nucleotide sequences disclosed inthe US Patent Application Publication No. 20110167520 A1, which isincorporated herein by reference in its entirety, or the complementsthereof.

The present invention also provides a computer readable medium havingrecorded thereon one or more of the nucleotide sequences encoding aprotein or fragment thereof, wherein the amino acid sequence of theprotein or fragment thereof is one of those amino acid sequencesdisclosed in the aforementioned US Patent Application Publication No.20110167520 A1.

The present invention also provides a method for using the computermedia of the present invention in isolating/identifying nucleic acidsencoding insect inhibitory proteins, or proteins involved inbiosynthesis of antibiotics.

A specific Xenorhabdus species, Xs85816, deposited according to theBudapets Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures with the AgricultureResearch Culture Collection (NRRL) International Depositary Authority at1815 North University Street, in Peoria, Ill. ZIP 61604 U.S.A. on Jun.22, 2000 and designated as NRRLB-30306, exhibiting insecticidal activityagainst piercing and sucking insects and against boll weevil iscontemplated as a source for DNA sequences encoding insecticidalproteins, and when formulated into a composition of matter as a spray,powder or emulsion, for the treatment of plants or animals to inhibitinsect infestation.

Another aspect of the present invention provides a method for isolatingnovel Xenorhabdus and Photorhabdus insect inhibitory species and theirsymbiont specific host entomopathogenic nematodes, wherein anentomopathogenic nematode containing sample of leaf litter, soil, orother earth derived organic sample is collected, and infested with oneor more insect larvae. Insect larvae used for such isolation should bepreferred target insect larvae commonly associated with insectinfestation or pest pressure. The insect infested sample should beincubated for a period of time in order to allow for nematode ingressinto the body(ies) of the target insect larvae, and in order for thesymbiont Xenorhabdus or Photorhabdus insect inhibitory bacteria to bereleased into the contents of the larvae, and for a period of time suchthat the bacteria proliferate within the body of the larvae. Preferably,the insect larvae are growth inhibited by the infestation, and morepreferably, the infested larvae are killed by the infestation of theentomopathogenic compositions produced from either the nematode or fromthe symbiont bacteria, or both. The bacteria are subsequently isolatedand purified using established culture methods, and the nematode hostscan be similarly isolated and purified, and both isolates can bemaintained using various means known in the art. Preferably, theentomopathogenic nematodes selectively target specific insect larvae,and more preferably specific strains of insect larvae, such that anygiven entomopathogenic nematode species is attracted preferentially tothat category and class of insect, and preferably the symbiont insectinhibitory bacterium released into the selectively targeted specificinsect larvae body is capable of overwhelming the insect larvae so as toelicit growth inhibition, feeding inhibition, death, or a combination ofthese effects. Evidence of these effects can be verified by selectivelyproviding isolated and purified bacteria obtained from suchentomopathogenic nematode infections, bacterial cell extracts, orculture extracts or compositions, to insect larvae in various types ofbioassays, followed by monitoring of the insect larvae in comparison tocontrol larvae in order to establish effective insect inhibition.

Yet another aspect of the present invention is the provision of a kitfor isolating novel entomopathogenic nematodes and their symbiont insectinhibitory bacteria.

Additionally, a novel method for assaying Xenorhabdus or Photorhabdusspecies for their insect inhibitory effects upon piercing and suckinginsects, including Lygus species, is provided comprising providing anentomopathogenic nematode composition to such an insect comprising aXenorhabdus species or a Photorhabdus species wherein said compositionis ingested by said insect through a saculus bounded on one side nearestand accessible to the insect by an insect probosis penetrable membraneand on the opposite side of said insect probosis penetrable membrane bya fluid reservoir, said reservoir forming a single layer, said layerhaving a first and a second surface, said first surface contacting saidinsect probosis penetrable membrane on the fluid side of the membrane,and said second surface contacting the first surface of a semipermeablemembrane having two surfaces, wherein said semipermeable membranerestricts the molecules which are greater than about 100 K Da, greaterthan about 115 K Da, greater than about 120 k Da, and/or greater thanabout 130 k Da, from entry into said fluid reservoir, and wherein saidsecond surface of said semipermeable membrane is contacted by a secondfluid reservoir containing said molecules; the inhibition of growth orviability of the insect being monitored upon the ingestion of saidmolecules by the insect through its probosis.

Another aspect of the present invention is provided by a method foridentifying an insect inhibitory protein produced from a Xenorhabdus ora Photorhabdus species which is specific for inhibiting a particulargenus or species of insect larvae. The method consists of exposing afirst larvae to a particular genus or species of nematode containingeither a Xenorhabdus or a Photorhabdus species of bacterium so that thenematode is allowed to invade the larvae, and release the bacteria fromits gut into the insect larvae haemolymphe. The bacteria are allowed toproliferate for a period of time, generally two or so days, and aresubsequently harvested from the larvae heamolymphe and isolated by purecultured onto indicator agar or other medium selective foridentification of the bacterial strain. The bacterial strain are thenstored for further use using a variety of means available in the art.The pure cultured bacterial strain is then grown in a specified medium,and the medium is harvested and filtered through a sub-micron filter toeliminate the presence of the bacterial cells. The protein profile ofthe filtered medium is then analyzed by a number of means known in theart and compared to the protein profile of medium harvested from thesame pure culture of bacteria which were passaged through thehaemolymphe of another genus or species of insect larvae. Differentproteins appearing in the profile of the medium from the bacteriapassaged through the haemolymphe of another genus or species of insectlarvae are then isolated and purified. The isolated and purifiedprotein(s) can then be used to produce antibodies directed to theprotein(s), said antibodies being useful for identifying genomic DNAclones which express all or a portion of the target protein to whichantibodies were raised. Alternatively, N or C-terminal sequence data canbe obtained which would allow for the production of redundantoligonucleotides for use as probes to identify genomic DNA sequencesobtained from the bacterium which hybridize to the oligonucleotides,presumably also being genomic DNA sequences encoding the protein(s)identified as being different in the profile analysis from the originalbacterial strains' profiled proteins. Such proteins, and the genesencoding these proteins, are candidate for being the proteins and genesencoding these proteins which are insect inhibitory and insect speciesspecific.

It is also conceived herein that the nematode may produce proteins orother accessory factors which, when combined with the particularsymbiont bacteria, trigger, activate, or enhance the expression of theproper insect specific insect inhibitory protein(s).

A further embodiment of the invention provides a method for selectingone or more insect inhibitory protein exhibiting enhanced insectinhibitory properties directed to the control of a selected insect pestspecies from a Xenorhabdus or a Photorhabdus bacterium species. Themethod consists of infesting an insect pest larvae with an insectpathogenic nematode. The nematode infestation results in the productionof juvenile nematodes obtained from the infested insect pest, and aXenorhabdus or a Photorhabdus bacterium species isolated and purifiedfrom the haemolymphe of the infested insect pest larvae. The isolatedand purified bacterium species is grown in liquid broth culture, and theculture broth is isolated and purified away from the bacterium species.The broth is tested for the presence of an insect inhibitorycomposition. The composition is identified, purified and characterizedas one or more proteins. Each protein is used to produce antibodies orN-terminal sequence information. The antibodies are used to screen arecombinant genomic library produced from the genome of the isolated andpurified bacterium species, resulting in the identification of one ormore clones expressing the protein. The one or more clones expressingthe protein can be partially sequenced to obtain DNA sequenceinformation that can be used to scan the genomic library disclosedherein, allowing one or more loci corresponding to the partial sequencewithin the genome of the bacterium to be identified, as well as thecomplete coding sequence of the gene encoding the protein exhibitingenhanced insect inhibitory properties directed to the control of aselected insect pest species. The N-terminal sequence is used to screenfor the presence of genomic nucleotide sequences encoding the N-terminalsequence from the genomic library described herein, which is used toobtain the full length sequence of the gene encoding the proteinexhibiting enhanced insect inhibitory properties directed to the controlof a selected insect pest species.

Alternatively, the nematode/bacterial symbiont pair can be infested intotwo or more different insect pests, the bacterial symbiont from thehaemolymphe of each insect pest harvested, isolated and purified, andthen each isolate grown in liquid broth culture to produce anextracellular protein profile determinable by size exclusionchromotography, SDS-PAGE, two dimensional gel electrophoresis, or asuitable method known in the art to identify protein or proteinsproduced by one isolate harvested from one insect pest which are notproduced by another isolate harvested from a different pest. The gene orgenes encoding the protein or proteins can be identified as describedabove.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention it has been discovered that thefollowing compositions comprising Xenorhabdus species and Photorhabdusspecies of bacteria commonly symbiotically associated with insectpathogenic nematodes, the individual nematodes, and the nucleic acidsand amino acids encoded by the said nucleic acids derived from thesebacteria and their host nematodes are surprisingly useful in providingcompositions comprising insect inhibitory proteins, proteins capable ofconferring antibiotic resistance, microbial inhibitory proteinsincluding bactericidal, bacteriostatic, fungicidal, and fungistaticproteins, proteins capable of conferring resistance to heavy metals orother toxic compositions, proteins and compositions capable ofconferring pharmaceutical advantages such as antineoplastic, acaricidal,anti-inflammatory and anti-ulcerogenic properties, polyketide synthases,transposons and mobile genetic elements and their correspondingtransposases, excisases, integrases, and invertases, phage and phageparticle proteins, other useful proteins homologous to proteins derivedfrom Xenorhabdus, Photorhabdus, Serratia, Yersinia, Salmonella, E. coli,and Erwinia sp. among others, ribosomal RNA (rRNA), and transfer RNA(tRNA). In addition, antibodies directed to the above mentioned proteinsand fragments thereof have been discovered to be of particular utilityin the present invention. The invention also relates to methods of usingthe disclosed nucleic acid molecules, proteins, fragments of proteins,and antibodies, for example, for gene identification and analysis,preparation of constructs, transformation of cells with nucleotidecompositions disclosed herein to produce Xenorhabdus or Photorhabdusproteins or fragments thereof, in particular novel insect inhibitory,bactericidal, fungicidal and nematicidal proteins.

Agents of the Present Invention

Nucleic Acid Molecules

One aspect of the present invention relates to an isolated nucleic acidmolecule having a nucleotide sequence, wherein: (1) the nucleotidesequence hybridized under stringent conditions to a second isolatednucleic acid molecule comprising SEQ ID NO:1 or the complements thereof;(2) the nucleotide sequence is a portion of SEQ ID NO:1; or (3) thenucleotide sequence is the complement of (1) or (2).

The term “nucleic acid” means a single or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Nucleic acids may also optionally contain synthetic, non-natural oraltered nucleotide bases that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid.

The term “an isolated nucleic acid” refers to a nucleic acid that is nolonger accompanied by some of materials with which it is associated inits natural state or to a nucleic acid the structure of which is notidentical to that of any of naturally occurring nucleic acid. Examplesof an isolated nucleic acid include: (1) DNAs which have the sequence ofpart of a naturally occurring genomic DNA molecules but are not flankedby two coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (2) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (3) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; (4)recombinant DNAs; and (5) synthetic DNAs. An isolated nucleic acid mayalso be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

The term “nucleotide sequence” refers to both the sense and antisensestrands of a nucleic acid as either individual single strands or in theduplex. It includes, but is not limited to, self-replicating plasmids,chromosomal sequences, and infectious polymers of DNA or RNA.

A nucleotide sequence is said to be the “complement” of anothernucleotide sequence if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the sequences is complementary to anucleotide of the other.

A “coding sequence” is a nucleotide sequence which is translated into apolypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, genomic DNA, cDNA, and recombinantpolynucleotide sequences.

The term “recombinant DNAs” refers to DNAs that contains a geneticallyengineered modification through manipulation via mutagenesis,restriction enzymes, and the like.

The term “synthetic DNAs” refers to DNAs assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form DNA segments which are then enzymatically assembled toconstruct the entire DNA. “Chemically synthesized”, as related to asequence of DNA, means that the component nucleotides were assembled invitro. Manual chemical synthesis of DNA may be accomplished using wellestablished procedures, or automated chemical synthesis can be performedusing one of a number of commercially available machines.

Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Appropriatestringent conditions are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6 (incorporated herein by reference). For the purposesof this disclosure, stringent conditions include at least one wash(usually 2) in 0.2×SSC at a temperature of at least about 50° C.,usually about 55° C., for 20 minutes, or equivalent conditions.

The hybridization portion of the two hybridizing nucleic acids isusually at least 40 nucleotides in length, more usually at least about75 nucleotides in length, more particularly at least 100 nucleotides inlengths. The hybridizing portion of the hybridizing nucleic acid is atleast 80%, at least 90%, or at least 98% identical to SEQ ID NO:1.

Another aspect of the present invention relates to an isolated nucleicacid molecule comprising one or more open reading frames listed in Table1 of the US Patent Application Publication No. 20110167520 A1, which isincorporated herein by reference in its entirety. An “open readingframe” (ORF) is a region of a nucleotide sequence which encodes apolypeptide. This region may represent a portion of a coding sequence ora total coding sequence. The table sets forth a list of open readingframes identified in the isolated nucleic acid molecules, wherein theopen reading frames encode Xenorhabdus proteins or polypeptide orfragments thereof which are homologues of known proteins or unknownproteins, or of tRNA's or rRNA's or fragments thereof which arehomologues of known tRNA's or rRNA's.

Open reading frames in genomic sequences can be screened for thepresence of protein homologues utilizing one or a number of differentsearch algorithms that have been developed, one example of which are thesuite of programs referred to as BLAST programs. There are fiveimplementations of BLAST, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology12:76-80 (1994); Birren et al., Genome Analysis 1:543-559 (1997)). Otherexamples of suitable programs that can be utilized are well known in theart. In addition, unidentified reading frames may be screened for bygene prediction software such as GenScan, which is located athttp://gnomic.stanford.edu/GENSCANW.html. Novel genes, i.e., with noknown homologs, can be predicted with the program GeneMark, whichcalculates the probability of a gene based on the presence of agene-like ‘grammar’ in the DNA sequence (i.e., start and stop signals,and a significant open reading frame) and statistical analyses ofprotein-coding potential through biases in putative codon usage (seehttp://genemark.biology.gatech.edu/GeneMark for details).

The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence, wherein: (1) the nucleotide sequencehybridizes under stringent conditions to a second isolated nucleic acidmolecule, wherein the hybridizing portion of the nucleotide sequence ofthe second isolated nucleic acid molecule encodes a polypeptide orprotein having an amino acid sequence of SEQ ID NO:2; (2) the nucleotidesequence encodes a polypeptide or protein, wherein the amino acidsequence of the polypeptide or protein is substantially identical to SEQID NO:2; or (3) the nucleotide sequence is the complement of (1) or (2).

In one embodiment, an isolated nucleic acid molecule comprises anucleotide sequence, wherein the nucleotide sequence encodes apolypeptide or protein having an amino acid sequence that issubstantially identical to SEQ ID NO:2.

The term “polypeptide” or “protein” refers to a linear polymer composedof amino acids connected by peptide bonds.

By “substantial identical” or “substantially identical” as used inreference to two amino acid sequences, it is meant that one amino acidsequence is identical to the other amino acid sequence or has at least50% sequence identity, at least 70% sequence identity, preferably atleast 80%, more preferably at least 90%, and most preferably at least95% identity when compared to the other amino acid sequence as areference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

Polypeptides which are “substantially similar” share sequences as notedabove except that residue positions which are not identical may differby conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. “Conservative amino acid substitutions” refer tosubstitutions of one or more amino acids in a native amino acid sequencewith another amino acid(s) having similar side chains, resulting in asilent change. Conserved substitutes for an amino acid within a nativeamino acid sequence can be selected from other members of the group towhich the naturally occurring amino acid belongs. For example, a groupof amino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine.

Optimal alignment of sequences for comparison can use any means toanalyze sequence identity (homology) known in the art, e.g., by theprogressive alignment method of termed “PILEUP” (Morrison, Mol. Biol.Evol. 14:428-441 (1997), as an example of the use of PILEUP); by thelocal homology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482(1981)); by the homology alignment algorithm of Needleman & Wunsch (J.Mol. Biol. 48:443 (1970)); by the search for similarity method ofPearson (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)); by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Geneprogram by Intelligenetics, Mountain View, Calif., described by, e.g.,Higgins, Gene 73: 237-244 (1988); Corpet, Nucleic Acids Res.16:10881-10890 (1988); Huang, Computer Applications in the Biosciences8:155-165 (1992); and Pearson, Methods in Mol. Biol. 24:307-331 (1994);Pfam (Sonnhammer, Nucleic Acids Res. 26:322-325 (1998); TreeAlign (Hein,Methods Mol. Biol. 25:349-364 (1994); MES-ALIGN, and SAM sequencealignment computer programs; or, by manual visual inspection.

Another example of algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul et al,J. Mol. Biol. 215: 403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/; see alsoZhang, Genome Res. 7:649-656 (1997) for the “PowerBLAST” variation. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence that eithermatch or satisfy some positive valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al, J. Mol. Biol.215: 403-410 (1990)). These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992)) alignments(B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. The term BLAST refers to the BLAST algorithm which performs astatistical analysis of the similarity between two sequences; see, e.g.,Karlin, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a nucleic acid is considered similar to areference sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about0.1, more preferably less than about 0.01, and most preferably less thanabout 0.001.

One skilled in the art will recognize that these values of sequenceidentity can be appropriately adjusted to determine correspondingsequence identity of two nucleotide sequences encoding the proteins ofthe present invention by taking into account codon degeneracy,conservative amino acid substitutions, reading frame positioning and thelike. Substantial identity of nucleotide sequences for these purposesnormally means sequence identity of at least 40%, preferably at least60%, more preferably at least 90%, and most preferably at least 95%.

The term “codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. The skilled artisan iswell aware of the “codon-bias” exhibited by a specific host cell inusage of nucleotide codons to specify a given amino acid. Therefore,when synthesizing a gene for expression in a host cell, it is desirableto design the gene such that its frequency of codon usage approaches thefrequency of preferred codon usage of the host cell.

The present invention also includes an isolated nucleic acid comprisinga nucleotide sequence encoding a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2 with conservative amino acidsubstitutions.

In a preferred embodiment of the present invention, the isolated nucleicacid molecule comprising a nucleotide sequence encodes an insectinhibitory protein, wherein the nucleotide sequence is SEQ ID NO:1. Theterm “insect inhibitory protein” refers to any polypeptide or protein ora substantial portion thereof that exhibits insect inhibitory activity,wherein said activity is manifested by inhibiting the growth ordevelopment of, or contributing substantially to, or causing the deathof a Coleopteran, a Dipteran, a Lepidopteran, a Hemipteran, aHymenopteran, or a sucking and piercing insect or insect larvae thereof.For instance, the present invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding all or substantialportion of a polypeptide having an amino acid sequence substantiallyidentical to SEQ ID NO:2.

The term “insect inhibitory protein” also refers to any polypeptide orprotein with modified amino acid sequence, such as sequence which hasbeen mutated, truncated, increased and the like and which maintains atleast the insect inhibitory activity associated with the native protein.Accordingly, the isolated nucleic acids encoding those polypeptide orprotein with such modification are also within the scope of the presentinvention.

Another aspect of the present invention relates to a class of isolatednucleic acid molecules comprising promoter sequences or regulatoryelements, particularly those found within SEQ ID NO:1 or the complementthereof.

The term “promoter sequence” means a nucleotide sequence that is capableof, when located in cis to a structural nucleotide sequence encoding apolypeptide or protein, functioning in a way that directs expression ofone or more mRNA molecules that encodes the polypeptide or protein. Suchpromoter regions are typically found upstream of the trinucleotide ATGsequence at the start site of a protein coding region. Promotersequences can also include sequences from which transcription oftransfer RNA (tRNA) or ribosomal RNA (rRNA) sequences are initiated.Transcription involves the synthesis of an RNA chain representing onestrand of a DNA duplex. By “representing” it is meant that the RNA isidentical in sequence with one strand of the DNA; it is complementary tothe other DNA strand, which provides the template for its synthesis.Transcription takes place by the usual process of complementary basepairing, catalyzed and scrutinized by the enzyme RNA polymerase. Thereaction can be divided into three stages described as initiation,elongation and termination. Initiation begins with the binding of RNApolymerase to the double stranded (DS or ds) DNA. The sequence of DNArequired for the initiation reaction defines the promoter. The site atwhich the first nucleotide is incorporated is called the startsite orstartpoint of transcription. Elongation describes the phase during whichthe enzyme moves along the DNA and extends the growing RNA chain.Elongation involves the disruption of the DNA double stranded structurein which a transiently unwound region exists as a hybrid RNA-DNA duplexand a displaced single strand of DNA. Termination involves recognitionof the point at which no further bases should be added to the chain. Toterminate transcription, the formation of phosphodiester bonds mustcease and the transcription complex must come apart. When the last baseis added to the RNA chain, the RNA-DNA hybrid is disrupted, the DNAreforms into a duplex state, and the RNA polymerase enzyme and RNAmolecule are both released from the DNA. The sequence of DNA requiredfor the termination reaction is called the terminator.

Generally, for bacteria the optimal promoter is a sequence consisting ofa −35 hexamer separated by about 17 base pairs from a −10 hexamer andlies from about 7 to about 10 base pairs upstream of the startpoint oftranscription, but these sequences can vary among and between sequenceswhich are recognized by the RNA polymerase. The startpoint oftranscription generally lies from about 20 to about 50 base pairsupstream of the startpoint of translation of one or more open readingframes which comprise the entire length of an mRNA transcript. Somepromoters can be recognized by RNA polymerase alone and in these cases,an accessible promoter will always be transcribed. Promoter availabilitymay be determined by extraneous proteins, which either may act directlyat the promoter to block access by RNA polymerase, or may functionindirectly by controlling the structure of the genome in the region.Other promoters are not by themselves adequate to support transcriptioninitiation and thus ancillary protein and or RNA factors are required tofurther initiation. The additional protein or RNA factors usually act byrecognizing sequences of DNA that are close to, or overlap with, thesequence bound by RNA polymerase itself. Additionally, some of theseancillary factors must touch and concern the RNA polymerase in order toeffect efficient transcription initiation as well as transcriptionelongation.

Promoters of the present invention can be included within sequences upto 10 kb upstream of the trinucleotide ATG sequence at the start site ofa protein coding region, tRNA, or rRNA. Promoters of the presentinvention can preferably be included within sequences up to 5 kbupstream of the trinucleotide ATG sequence at the start site of aprotein coding region, tRNA or rRNA. Promoters of the present inventioncan more preferably be included within sequences up to 2 kb upstream ofthe trinucleotide ATG sequence at the start site of a protein codingregion, tRNA or rRNA. Promoters of the present invention can mostpreferably be included within sequences up to 500 bp upstream of thetrinucleotide ATG sequence at the start site of a protein, tRNA, or rRNAcoding region. While in many circumstances a 300 bp promoter may besufficient for expression, additional sequences may act to furtherregulate expression, for example, in response to biochemical,developmental or environmental signals. In a preferred embodiment of thepresent invention, the promoter is upstream of an nucleic acid sequencethat encodes a Xenorhabdus protein or fragment thereof.

The term “regulatory element” is intended to mean a series ofnucleotides that determines if, when, and at what level a particulargene is expressed. Regulatory DNA sequences specifically interact withregulatory or other proteins. Many regulatory elements act in cis (“ciselements”) and are believed to affect DNA topology, producing localconformations that selectively allow or restrict access of RNApolymerase to the DNA template or that facilitate selective opening ofthe double helix at the site of transcriptional initiation, i.e., thetranscriptional startsite referred to above. Cis elements occur within,near to, adjacent to, or at a distance from a particular promoter, butremain linked to the promoter sequence along the sequence ofphosphodiester bonds which comprise the nucleotide sequence within whichthe promoter resides. Cis elements are not limited to promoters, but maybe imparted to RNA sequences derived from transcription from DNAsequences of the present invention, wherein such RNA cis elements areinvolved in post transcriptional regulation of gene expression. Forexample, elements which are known as inverted repeat sequences canassist in the formation of hairpin structures which prevent, inhibit, orotherwise modulate the translational efficiency of the RNA sequence, orwhich regulate the survival of the RNA sequence. Other elements mayfunction to bind ribosomes or components which enhance or suppresstranslational efficiency. Cis elements can be identified using known ciselements as a target sequence or target motif in the BLAST. Promoters ofthe present invention include homologues of cis elements known to effectgene regulation that show homology with the nucleic acid molecules ofthe present invention.

The isolated nucleic acid molecules of the present invention alsoinclude nucleic acid molecules that encode ribosomal RNA (rRNA),transfer RNA (tRNA) molecules, or other nucleic acid molecules whichfunction to regulate gene expression, transcription, translation byacting alone or in combination with other cellular components inactivating, inhibiting, terminating or anti-terminating gene expressionfunctions, or by acting alone or in combination with other structuralmolecules to form or assist in the formation of said structuralmolecules.

A “substantial portion” of a nucleotide sequence comprises enough of thesequence to afford specific identification and/or isolation of a nucleicacid fragment comprising the sequence. In general, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

It is also contemplated by the inventors that the isolated nucleic acidmolecules of the present invention also include known types ofmodifications, for example, labels which are known in the art,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog. Other known modifications includeinternucleotide modifications, for example, those with unchargedlinkages (methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,proteins (including nucleases, metabolic toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (acridine,psoralen, etc.), those containing chelators (metals, radioactive metals,boron, oxidative metals, etc.), those containing alkylators, and thosewith modified linkages (alpha anomeric nucleic acids, etc.).

The nucleic acids of the present invention may be used to isolatenucleic acids encoding homologous proteins from the same or otherspecies. Isolation of homologous genes using sequence-dependentprotocols is well known in the art. Examples of sequence-dependentprotocols include, but are not limited to, methods of nucleic acidhybridization, and methods of DNA and RNA amplification as exemplifiedby various uses of nucleic acid amplification technologies (e.g.,polymerase chain reaction, ligase chain reaction).

For example, genes encoding homologous proteins, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe nucleic acids of the present invention as DNA hybridization probesto screen cDNA or genomic libraries from any desired organism employingmethodology well known to those skilled in the art. Methods for formingsuch libraries are well known in the art. Specific oligonucleotideprobes based upon the nucleic acids of the present invention can bedesigned and synthesized by methods known in the art. Moreover, theentire sequences of the nucleic acids can be used directly to synthesizeDNA probes by methods known to the skilled artisan such as random primerDNA labeling, nick translation, or end-labeling techniques, or RNAprobes using available in vitro transcription systems. In addition,specific primers can be designed and used to amplify a part or all ofthe sequences. The resulting amplification products can be labeleddirectly during amplification reactions or labeled after amplificationreactions, and used as probes to isolate full length cDNA or genomicDNAs under conditions of appropriate stringency.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance, thedisclosed nucleic acids may be used to define a pair of primers that canbe used with the polymerase chain reaction (Mullis, et al., Cold SpringHarbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al., EP 50,424;EP 84,796, EP 258,017, EP 237,362; Mullis, EP 201,184; Mullis et al.,U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki, R.et al., U.S. Pat. No. 4,683,194) to amplify and obtain any desirednucleic acid or fragment directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes.

In addition, two short segments of the nucleic acids of the presentinvention may be used in polymerase chain reaction protocols, forexample, the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. USA85:8998 (1988)), to amplify longer nucleic acids encoding homologousgenes from DNA or RNA from other sources.

Nucleic acids of interest may also be synthesized, either completely orin part, especially where it is desirable to provide plant-preferredsequences, by well-known techniques as described in the technicalliterature. See, e.g., Carruthers et al., Cold Spring Harbor Symp.Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc.105:661 (1983). Thus, all or a portion of the nucleic acids of thepresent invention may be synthesized using codons preferred by aselected plant host. Plant-preferred codons may be determined, forexample, from the codons used most frequently in the proteins expressedin a particular plant host species. Other modifications of the genesequences may result in mutants having slightly altered activity.

Availability of the nucleotide sequences encoding Xenorhabdus proteinsfacilitates immunological screening of DNA expression libraries.Synthetic polypeptides representing portions of the amino acid sequencesof Xenorhabdus proteins may be synthesized. These polypeptides can beused to immunize animals to produce polyclonal or monoclonal antibodieswith specificity for polypeptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen expressionlibraries to isolate genes of interest (Lemer, Adv. Immunol. 36: 1(1984); Sambrook et al., Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, (1989)). It isunderstood that people skilled in the art are familiar with the standardresource materials which describe specific conditions and procedures forthe construction, manipulation and isolation of antibodies (see, forexample, Harlow and Lane, In Antibodies: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1988)).

Another aspect of the present invention relates to a method forobtaining a nucleic acid comprising a nucleotide sequence encoding aXenorhabdus protein homologue having an amino acid sequence at least 70%identical to SEQ ID NO:2. In a preferred embodiment, the method of thepresent invention for obtaining a nucleic acid encoding all or asubstantial portion of the amino acid sequence of a Xenorhabdus proteinhomologue comprising: (a) probing an expression library with ahybridization probe comprising a nucleotide sequence encoding apolypeptide having an amino acid sequence set forth in SEQ ID NO:2; oran amino acid sequence set forth in SEQ ID NO:2 with conservative aminoacid substitutions; (b) identifying a DNA clone that hybridizes to thehybridization probe; (c) isolating the DNA clone identified in step (b);and (d) sequencing the DNA fragment that comprises the clone isolated instep (c) wherein the sequenced nucleic acid molecule encodes all or asubstantial portion of the amino acid sequence of the Xenorhabdusprotein homologue.

In another preferred embodiment, the method of the present invention forobtaining a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a Xenorhabdus protein homologue comprising: (a)synthesizing a first and a second oligonucleotide primers correspondingto a portion of the coding sequence of a second nucleic acid moleculeset forth in SEQ ID NO:1; and (b) amplifying a DNA insert present in acloning vector using the first and second oligonucleotide primers ofstep (a) wherein the amplified nucleic acid molecule encodes all or asubstantial portion of the amino acid sequence of the Xenorhabdusprotein homologue.

Protein and Polypeptide Molecules

The present invention, in another aspect, provides a substantiallypurified protein or polypeptide molecule comprising an amino acidsequence, wherein the amino acid sequence is defined as follows: (1) theamino acid sequence is encoded by a first nucleotide sequence whichspecifically hybridizes to the complement of a second nucleotidesequence set forth in SEQ ID NO:1; (2) the amino acid sequence isencoded by a third nucleotide sequence that is at least 50% identical toall or a substantial portion of a coding sequence located within SEQ IDNO:1; or (3) the amino acid sequence is substantially identical to SEQID NO:2. In alternative embodiments, the third nucleotide sequence is atleast 55% identical, at least 60% identical, at least 65% identical, atleast 70% identical, at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical toall or a substantial portion of a coding sequence located within SEQ IDNO:1. In a preferred embodiment, the third nucleotide sequence is 100%identical to all or a substantial portion of a coding sequence locatedwithin SEQ ID NO:1. In a preferred embodiment, the amino acid sequenceis at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, at least 95% identical, at least 98%identical to SEQ ID NO:2.

The term “substantially purified protein or polypeptide molecule” refersto a protein or polypeptide molecule separated from substantially allother molecules normally associated with it in its native state. Morepreferably a substantially purified protein or polypeptide molecule isthe predominant species present in a preparation. A substantiallypurified molecule may be greater than 60% free, preferably 75% free,more preferably 90% free, and most preferably 95% free from the othermolecules (exclusive of solvent) present in the natural mixture.

It is well known in the art that proteins or polypeptides may undergomodification, including post-translational modifications, such as, butnot limited to, disulfide bond formation, glycosylation,phosphorylation, or oligomerization. Thus, as used herein, the term“protein molecule” or “polypeptide molecule” includes any proteinmolecule that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids. This definition is meant to include norleucine,ornithine, homocysteine, and homoserine.

The polypeptides or proteins of the present invention may be producedvia chemical synthesis, or more preferably, by expression in a suitablebacterial or eukaryotic host. Suitable methods for expression aredescribed by Sambrook, et al., (In: Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)), or similar texts.

The polypeptides or protein molecule of the present invention may alsoinclude fusion protein or polypeptide molecules. A protein orpolypeptide molecule that comprises one or more additional polypeptideregions not derived from that protein molecule is a “fusion” protein orpolypeptide molecule. Such molecules may be derivatized to containcarbohydrate or other moieties (such as keyhole limpet hemocyanin,etc.). Fusion protein or polypeptide molecules of the present inventionare preferably produced via recombinant means.

The protein or polypeptide molecules of the present invention may alsoinclude protein or polypeptide molecules encoded by all or a substantialportion of protein-encoding sequences in SEQ ID NO:1 or complementsthereof or, fragments or fusions thereof in which conservative,non-essential, or not relevant, amino acid residues have been added,replaced, or deleted. An example of such a homologue is the homologueprotein from different strains or species. Such a homologue can beobtained by any of a variety of methods. For example, as indicatedabove, one or more of the disclosed sequences (all or a substantialportion of the protein-encoding sequence of SEQ ID NO:1 or complementsthereof) will be used to define a pair of primers that may be used toisolate the homologue-encoding nucleic acid molecules from any desiredspecies. Such molecules can be expressed to yield homologues byrecombinant means.

Antibodies

Another aspect of the present invention concerns antibodies,single-chain antigen binding molecules, or other proteins thatspecifically bind to one or more of the protein or polypeptide moleculesof the present invention and their homologues, fusions or fragments.Such antibodies may be used to quantitatively or qualitatively detectthe protein or polypeptide molecules of the present invention. As usedherein, an antibody or polypeptide is said to “specifically bind” to aprotein or polypeptide molecule of the present invention if such bindingis not competitively inhibited by the presence of non-related molecules.In a preferred embodiment the antibodies of the present invention bindto protein or polypeptide molecules of the present invention, in a morepreferred embodiment of the antibodies of the present invention bind toprotein or polypeptide molecules derived from Xenorhabdus.

Nucleic acid molecules that encode all or part of the protein orpolypeptide of the present invention can be expressed, via recombinantmeans, to yield protein or polypeptides that can in turn be used toelicit antibodies that are capable of binding the expressed protein orpolypeptide. Such antibodies may be used in immunoassays for thatprotein or polypeptide. Such protein or polypeptide-encoding molecules,or their fragments may be “fusion” molecules (i.e., a part of a largernucleic acid molecule) such that, upon expression, a fusion protein isproduced. It may be desirable to derivatize the obtained antibodies, forexample with a ligand group (such as biotin) or a detectable markergroup (such as a fluorescent group, a radioisotope or an enzyme). Suchantibodies may be used in immunoassays for that protein. In a preferredembodiment, such antibodies can be used to screen DNA expressionlibraries to isolate clones containing full-length insert of genes(Lemer, Adv. Immunol. 36: 1 (1984); Sambrook et al., Molecular Cloning:A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold SpringHarbor, (1989)).

The antibodies that specifically bind proteins and protein fragments ofthe present invention may be polyclonal or monoclonal, and may compriseintact immunoglobulins, or antigen binding portions of immunoglobulins(such as (F(ab′), F(ab′)2 fragments), or single-chain immunoglobulinsproducible, for example, via recombinant means). It is understood thatpractitioners are familiar with the standard resource materials whichdescribe specific conditions and procedures for the construction,manipulation and isolation of antibodies (see, for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1988)).

In a preferred embodiment, the antibodies of the present inventionspecifically bind to one or more of the insect inhibitory polypeptidesor proteins of the present invention. Such antibodies may be used todetect the presence of such insect inhibitory polypeptides or proteinsin a sample.

The present invention also provide a method for detecting an insectinhibitory polypeptide or protein in a biological sample, the methodgenerally comprising: (1) obtaining a biological sample; (2) contactingthe sample with an antibody that specifically binds to the polypeptideor protein, under conditions effective to allow the formation ofcomplexes; and (3) detecting the complexes so formed.

Plant Constructs and Plant Transformants

The present invention also relates to a plant recombinant vector orconstruct comprising a structural nucleotide sequence encoding aXenorhabdus protein or polypeptide. In a preferred embodiment, a plantrecombinant vector or construct of the present invention comprises astructural nucleotide sequence encoding an insect inhibitory protein orpolypeptide of the present invention. The present invention also relatesto a transformed plant cell or plant comprising in its genome anexogenous nucleic acid encoding one or more Xenorhabdus or Photorhabdusproteins or polypeptides of the present invention. The present inventionalso relates to methods for creating a transgenic plant in which one ormore Xenorhabdus or Photorhabdus proteins or polypeptides of the presentinvention are overexpressed.

By “exogenous” it is meant that a nucleic acid originates from outsidethe plant. An exogenous nucleic acid can have a naturally occurring ornon-naturally occurring nucleotide sequence. One skilled in the artunderstands that an exogenous nucleic acid can be a heterologous nucleicacid derived from a different plant species than the plant into whichthe nucleic acid is introduced or can be a nucleic acid derived from thesame plant species as the plant into which it is introduced.

The term “overexpression” refers to the expression of a polypeptide orprotein encoded by an exogenous nucleic acid introduced into a hostcell, wherein said polypeptide or protein is either not normally presentin the host cell, or wherein said polypeptide or protein is present insaid host cell at a higher level than that normally expressed from theendogenous gene encoding said polypeptide or protein. By “endogenousgene” refers to a native gene in its natural location in the genome ofan organism.

The term “genome” as it applies to plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components of the cell. DNAs of the present inventionintroduced into plant cells can therefore be either chromosomallyintegrated or organelle-localized. The term “genome” as it applies tobacteria encompasses both the chromosome and plasmids within a bacterialhost cell. Encoding DNAs of the present invention introduced intobacterial host cells can therefore be either chromosomally integrated orplasmid-localized.

Method which are well known to those skilled in the art may be used toconstruct the plant recombinant construct or vector of the presentinvention. These method include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y. (1989).

A plant recombinant construct or vector of the present inventioncontains a structural nucleotide sequence encoding one or moreXenorhabdus or Photorhabdus proteins or polypeptides of the presentinvention and operably linked regulatory sequences or control elements.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the operably linked structuralnucleotide sequence. “Expression” refers to the transcription and stableaccumulation of sense or antisense RNA derived from the nucleic acid ofthe present invention. Expression may also refer to translation of mRNAinto a polypeptide or protein. “Sense” RNA refers to RNA transcript thatincludes the mRNA and so can be translated into protein by the cell.“Antisense RNA” refers to a RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target gene (U.S. Pat. No. 5,107,065, incorporatedherein by reference). The complementarity of an antisense RNA may bewith any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-translated sequence, introns, or the codingsequence. “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA.

“Regulatory sequences” or “control elements” refer to nucleotidesequences located upstream (5′ noncoding sequences), within, ordownstream (3′ non-translated sequences) of a structural nucleotidesequence, and which influence the transcription, RNA processing orstability, or translation of the associated structural nucleotidesequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

The promoter sequence may consist of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence which can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions.

Promoters which are known or are found to cause transcription of DNA inplant cells can be used in the present invention. Such promoters may beobtained from a variety of sources such as plants and plant viruses. Anumber of promoters, including constitutive promoters, induciblepromoters and tissue-specific promoters, that are active in plant cellshave been described in the literature. It is preferred that theparticular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount of aprotein to cause the desired phenotype. In addition to promoters thatare known to cause transcription of DNA in plant cells, other promotersmay be identified for use in the current invention by screening a plantcDNA library for genes that are selectively or preferably expressed inthe target tissues and then determine the promoter regions.

The term “constitutive promoter” means a regulatory sequence whichcauses expression of a structural nucleotide sequence in most cells ortissues at most times. Constitutive promoters are active under mostenvironmental conditions and states of development or celldifferentiation. A variety of constitutive promoters are well known inthe art. Examples of constitutive promoters that are active in plantcells include but are not limited to the nopaline synthase (NOS)promoters; the cauliflower mosaic virus (CaMV) 19S and 35S; the tobaccomosaic virus promoter; the figwort mosaic virus promoters; and actinpromoters, such as the Arabidopsis actin gene promoter (see, e.g.,Huang, Plant Mol. Biol. 33:125-139 (1997)).

The term “inducible promoter” refers to a regulatory sequence whichcauses conditional expression of a structural nucleotide sequence underthe influence of changing environmental conditions or developmentalconditions. Examples of inducible promoters include but are not limitedto the light-inducible promoter from the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO); thedrought-inducible promoter of maize (Busk, Plant J. 11:1285-1295(1997)); the cold, drought, and high salt inducible promoter from potato(Kirch, Plant Mol. Biol. 33:897-909 (1997)); a nitrate-induciblepromoter derived from the spinach nitrite reductase gene (Back et al.,Plant Mol. Biol. 17:9 (1991)); salicylic acid inducible promoter (Ukneset al., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245(1995)); the auxin-response elements El promoter fragment (AuxREs) inthe soybean (Glycine max L.) (Liu, Plant Physiol. 115:397-407 (1997));the auxin-responsive Arabidopsis GST6 promoter (also responsive tosalicylic acid and hydrogen peroxide) (Chen, Plant J. 10: 955-966(1996)); the auxin-inducible parC promoter from tobacco (Sakai,37:906-913 (1996)); a plant biotin response element (Streit, Mol. PlantMicrobe Interact. 10:933-937 (1997)); the promoter responsive to thestress hormone abscisic acid (Sheen, Science 274:1900-1902 (1996)); themaize In2-2 promoter activated by benzenesulfonamide herbicide safeners(De Veyl der, Plant Cell Physiol. 38:568-577 (1997)); atetracycline-inducible promoter, such as the promoter for the Avenasativa L. (oat) arginine decarboxylase gene (Masgrau, Plant J.11:465-473 (1997)); and a salicylic acid-responsive element (Stange,Plant J. 11:1315-1324 (1997)).

The term “tissue-specific promoter” means a regulatory sequence thatcauses transcriptions or enhanced transcriptions of DNA in specificcells or tissues at specific times during plant development, such as invegetative tissues or reproductive tissues. Examples of tissue-specificpromoters under developmental control include promoters that initiatetranscription only (or primarily only) in certain tissues, such asvegetative tissues, e.g., roots, leaves or stems, or reproductivetissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or anyembryonic tissue. Reproductive tissue specific promoters may be, e.g.,ovule-specific, embryo-specific, endosperm-specific,integument-specific, seed coat-specific, pollen-specific,petal-specific, sepal-specific, or some combination thereof. One ofskill will recognize that a tissue-specific promoter may driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but may also leadto some expression in other tissues as well.

A variety of promoters specifically active in vegetative tissues, suchas leaves, stems, roots and tubers, can also be used to express thenucleic acids of the invention. Examples of tuber-specific promotersinclude but are not limited to the class I and II patatin promoters(Bevan et al., EMBO J. 8: 1899-1906 (1986); Koster-Topfer et al., MolGen Genet. 219: 390-396 (1989); Mignery et al., Gene. 62: 27-44 (1988);Jefferson et al., Plant Mol. Biol. 14: 995-1006 (1990)), the promoterfor the potato tuber ADPGPP genes, both the large and small subunits;the sucrose synthase promoter (Salanoubat and Belliard, Gene. 60: 47-56(1987), Salanoubat and Belliard, Gene. 84: 181-185 (1989)); and thepromoter for the major tuber proteins including the 22 kd proteincomplexes and proteinase inhibitors (Hannapel, Plant Physiol. 101:703-704 (1993)). Examples of leaf-specific promoters include but are notlimited to the ribulose biphosphate carboxylase (RBCS or RuBISCO)promoters (see, e.g., Matsuoka, Plant J. 6:311-319 (1994)); the lightharvesting chlorophyll a/b binding protein gene promoter (see, e.g.,Shiina, Plant Physiol. 115-0.477-483 (1997); Casal, Plant Physiol.116:1533-1538 (1998)); and the Arabidopsis thaliana myb-related genepromoter (Atmyb5) (Li, FEBS Lett. 379:117-121 (1996)). Examples ofroot-specific promoter include but are not limited to the promoter forthe acid chitinase gene (Samac et al., Plant Mol. Biol. 25: 587-596(1994)); the root specific subdomains of the CaMV35S promoter that havebeen identified (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.)86:7890-7894 (1989)); the ORF13 promoter from Agrobacterium rhizogeneswhich exhibits high activity in roots (Hansen, Mol. Gen. Genet.254:337-343 (1997)); the promoter for the tobacco root-specific geneTobRB7 (Yamamoto, Plant Cell 3:371-382 (1991)); and the root cellspecific promoters reported by Conkling et al. (Conkling et al., PlantPhysiol. 93:1203-1211 (1990)).

Another class of useful vegetative tissue-specific promoters aremeristermatic (root tip and shoot apex) promoters. For example, the“SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in thedeveloping shoot or root apical meristems (Di Laurenzio, Cell 86:423-433(1996); Long, Nature 379:66-69 (1996)), can be used. Another example ofa useful promoter is that which controls the expression of3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whoseexpression is restricted to meristematic and floral (secretory zone ofthe stigma, mature pollen grains, gynoecium vascular tissue, andfertilized ovules) tissues (see, e.g., Enjuto, Plant Cell. 7:517-527(1995)). Also another example of a useful promoter is that whichcontrols the expression of knl-related genes from maize and otherspecies which show meristern-specific expression (see, e.g., Granger,Plant Mol. Biol. 31:373-378 (1996); Kerstetter, Plant Cell 6:1877-1887(1994); Hake, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51(1995). Another example of a meristematic promoter is the Arabidopsisthaliana KNAT1 promoter. In the shoot apex, KNAT1 transcript islocalized primarily to the shoot apical meristem; the expression ofKNATI in the shoot meristem decreases during the floral transition andis restricted to the cortex of the inflorescence stem (see, e.g.,Lincoln, Plant Cell 6:1859-1876 (1994)).

Suitable seed-specific promoters can be derived from the followinggenes: MAC1 from maize (Sheridan, Genetics 142:1009-1020 (1996); Cat3from maize (GenBank No. L05934, Abler, Plant Mol. Biol. 22:10131-1038(1993); vivparous-1 from Arabidopsis (Genbank No. U93215); Atimycl fromArabidopsis (Urao, Plant Mol. Biol. 32:571-57 (1996); Conceicao, Plant5:493-505 (1994); napA from Brassica napus (GenBank No. J02798); thenapin gene family from Brassica napus (Sjodahl, Planta 197:264-271(1995)).

The ovule-specific BEL1 gene described in Reiser (1995) Cell 83:735-742,GenBank No. U39944, can also be used. See also Ray (1994) Proc. Natl.Acad. Sci. USA 91:5761-5765. The egg and central cell specific FIEEIpromoter is also a useful reproductive tissue-specific promoter.

A maize pollen-specific promoter has been identified in maize (Guerrero(1990) Mol. Gen. Genet. 224:161-168). Other genes specifically expressedin pollen are described, e.g., by Wakeley (1998) Plant Mol. Biol.37:187-192; Ficker (1998) Mol. Gen. Genet. 257:132-142; Kulikauskas(1997) Plant Mol. Biol. 34:809-814; Treacy (1997) Plant Mol. Biol.34:603-611.

Promoters derived from genes encoding embryonic storage proteins, whichincludes the gene encoding the 2S storage protein from Brassica napus(Dasgupta, Gene 133:301-302 (1993); the 2 s seed storage protein genefamily from Arabidopsis; the gene encoding oleosin 20 kD from Brassicanapus (GenBank No. M63985); the genes encoding oleosin A (Genbank No.U09118) and oleosin B (Genbank No. U09119) from soybean; the geneencoding oleosin from Arabidopsis (Genbank No. Z17657); the geneencoding oleosin 18 kD from maize (GenBank No. J05212, Lee, Plant Mol.Biol. 26:1981-1987 (1994)); and the gene encoding low molecular weightsulphur rich protein from soybean (Choi, Mol Gen, Genet. 246:266-268(1995)), can also be used.

Promoters derived from genes encoding for zein genes (including the 15kD, 16 kD, 19 kD, 22 kD, 27 kD, and gamma genes) (Pedersen et al., Cell29: 1015-1026 (1982)) can be also used. The zeins are a group of storageproteins found in maize endosperm.

Other promoters known to function, for example, in maize, include thepromoters for the following genes: waxy, Brittle, Shrunken 2, Branchingenzymes I and II, starch synthases, debranching enzymes, oleosins,glutelins, and sucrose synthases. A particularly preferred promoter formaize endosperm expression is the promoter for the glutelin gene fromrice, more particularly the Osgt-1 promoter (Zheng et al., Mol. CellBiol. 13: 5829-5842 (1993), herein incorporated by reference in itsentirety). Examples of promoters suitable for expression in wheatinclude those promoters for the ADPglucose pyrophosphorylase (ADPGPP)subunits, the granule bound and other starch synthases, the branchingand debranching enzymes, the embryogenesis-abundant proteins, thegliadins, and the glutenins Examples of such promoters in rice includethose promoters for the ADPGPP subunits, the granule bound and otherstarch synthases, the branching enzymes, the debranching enzymes,sucrose synthases, and the glutelins. A particularly preferred promoteris the promoter for rice glutelin, Osgt-1. Examples of such promotersfor barley include those for the ADPGPP subunits, the granule bound andother starch synthases, the branching enzymes, the debranching enzymes,sucrose synthases, the hordeins, the embryo globulins, and the aleuronspecific proteins.

A tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(Blume, Plant J. 12:731-746 (1997)). Other exemplary promoters includethe pistol specific promoter in the potato (Solarium tuberosum L.) SK2gene, encoding a pistil-specific basic endochitinase (Ficker, Plant Mol.Biol. 35:425-431 (1997)); the Blec4 gene from pea (Pisum sativum cv.Alaska), active in epidermal tissue of vegetative and floral shootapices of transgenic alfalfa. This makes it a useful tool to target theexpression of foreign genes to the epidermal layer of actively growingshoots. The tissue specific E8 promoter from tomato is also useful fordirecting gene expression in fruits.

It is recognized that additional promoters that may be utilized aredescribed, for example, in U.S. Pat. Nos. 5,378,619, 5,391,725,5,428,147, 5,447,858, 5,608,144, 5,608,144, 5,614,399, 5,633,441,5,633,435, and 4,633,436, all of which are herein incorporated in theirentirety. In addition, a tissue specific enhancer may be used (Fromm etal., The Plant Cell 1:977-984 (1989), herein incorporated by referencein its entirety). It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, DNA fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-translated sequences” refer to DNA sequences locateddownstream of a structural nucleotide sequence and include sequencesencoding polyadenylation and other regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalfunctions in plants to cause the addition of polyadenylate nucleotidesto the 3′ end of the mRNA precursor. The polyadenylation sequence can bederived from the natural gene, from a variety of plant genes, or fromT-DNA. An example of the polyadenylation sequence is the nopalinesynthase 3′ sequence (NOS 3′; Fraley et al., Proc. Natl. Acad. Sci. USA80: 4803-4807 (1983)). The use of different 3′ non-translated sequencesis exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.

Generally, optimal expression in monocotyledonous and somedicotyledonous plants is obtained when an intron sequence is insertedbetween the promoter sequence and the structural gene sequence or,optionally, may be inserted in the structural coding sequence to providean interrupted coding sequence. An example of such an intron sequence isthe HSP 70 intron described in WO 93/19189.

A recombinant vector or construct of the present invention willtypically comprise a selectable marker which confers a selectablephenotype on plant cells. Selectable markers may also be used to selectfor plants or plant cells that contain the exogenous nucleic acidsencoding polypeptides or proteins of the present invention. The markermay encode biocide resistance, antibiotic resistance (e.g., kanamycin,G418 bleomycin, hygromycin, etc.), or herbicide resistance (e.g.,glyphosate, etc.). Examples of selectable markers include, but are notlimited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188(1985)) which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc.; a bar gene which codes for bialaphosresistance; a mutant EPSP synthase gene (Hinchee et al., Bio/Technology6:915-922 (1988)) which encodes glyphosate resistance; a nitrilase genewhich confers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) whichconfers imidazolinone or sulphonylurea resistance (European PatentApplication 154,204 (Sep. 11, 1985)); and a methotrexate resistant DHFRgene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405 (1987);Jefferson et al., EMBO J. 6:3901-3907 (1987)); an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta et al., Stadler Symposium11:263-282 (1988)); a β-lactamase gene (Sutcliffe et al., Proc. Natl.Acad. Sci. (U.S.A.) 75:3737-3741 (1978)), a gene which encodes an enzymefor which various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a luciferase gene (Ow et al., Science234:856-859 (1986)) a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci.(U.S.A.) 80:1101-1105 (1983)) which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikatu et al.,Bio/Technol. 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen.Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses tomelanin; an α-galactosidase, which will turn a chromogenic α-galactosesubstrate.

Included within the terms “selectable or screenable marker genes” arealso genes which encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin transferase), or proteins whichare inserted or trapped in the cell wall (such as proteins which includea leader sequence such as that found in the expression unit of extensionor tobacco PR-S). Other possible selectable and/or screenable markergenes will be apparent to those of skill in the art.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes which are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in K. Wising et al. Ann. Rev.Genetics, 22, 421 (1988), which is incorporated herein by reference.Preferred reporter genes include the beta-glucuronidase (GUS) of theuidA locus of E. coli, the chloramphenicol acetyl transferase gene fromTn9 of E. coli, the green fluorescent protein from the bioluminescentjellyfish Aequorea victoria, and the luciferase genes from fireflyPhotinus pyralis. An assay for detecting reporter gene expression maythen be performed at a suitable time after said gene has been introducedinto recipient cells. A preferred such assay entails the use of the geneencoding beta-glucuronidase (GUS) of the uidA locus of E. coli asdescribed by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) toidentify transformed cells.

In preparing the DNA constructs of the present invention, the variouscomponents of the construct or fragments thereof will normally beinserted into a convenient cloning vector, e.g., a plasmid that iscapable of replication in a bacterial host, e.g., E. coli. Numerousvectors exist that have been described in the literature, many of whichare commercially available. After each cloning, the cloning vector withthe desired insert may be isolated and subjected to furthermanipulation, such as restriction digestion, insertion of new fragmentsor nucleotides, ligation, deletion, mutation, resection, etc. so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

A recombinant vector or construct of the present invention may alsoinclude a chloroplast transit peptide, in order to target thepolypeptide or protein of the present invention to the plastid. The term“plastid” refers to the class of plant cell organelles that includesamyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts,etioplasts, leucoplasts, and proplastids. These organelles areself-replicating, and contain what is commonly referred to as the“chloroplast genome,” a circular DNA molecule that ranges in size fromabout 120 to about 217 kb, depending upon the plant species, and whichusually contains an inverted repeat region. Many plastid-localizedproteins are expressed from nuclear genes as precursors and are targetedto the plastid by a chloroplast transit peptide (CTP), which is removedduring the import steps. Examples of such chloroplast proteins includethe small subunit of ribulose-1,5-biphosphate carboxylase (ssRUBISCO,SSU), 5-enolpyruvateshikimate-3-phosphate synthase (EPSPS), ferredoxin,ferredoxin oxidoreductase, the light-harvesting-complex protein I andprotein II, and thioredoxin F. It has been demonstrated that non-plastidproteins may be targeted to the chloroplast by use of protein fusionswith a CTP and that a CTP sequence is sufficient to target a protein tothe plastid. Those skilled in the art will also recognize that variousother chimeric constructs can be made that utilize the functionality ofa particular plastid transit peptide to import the enzyme into the plantcell plastid depending on the promoter tissue specificity.

The present invention also provide a transgenic plant comprising in itsgenome an isolated nucleic acid which comprises: (A) a 5′ non-codingsequence which functions in the cell to cause the production of a mRNAmolecule; which is linked to (B) a structural nucleotide sequence,wherein the structural nucleotide sequence encodes a Xenorhabdus proteinor polypeptide of the present invention; which is linked to (C) a 3′non-translated sequence that functions in said cell to cause terminationof transcription. In a preferred embodiment, the amino acid sequence ofthe above described polypeptide or protein is substantially identical toSEQ ID NO:2.

The term “transgenic plant” refers to a plant that contains an exogenousnucleic acid, which can be derived from the same plant species or from adifferent plant species. Transgenic plants of the present inventionpreferably have incorporated into their genome or transformed into theirchloroplast or plastid genomes a selected polynucleotide (or“transgene”), that comprises at least a structural nucleotide sequencethat encodes an insect inhibitory polypeptide having an amino acidsequence as set forth in SEQ ID NO:2. Transgenic plants are also meantto comprise progeny (descendant, offspring, etc.) of any generation ofsuch a transgenic plant. A seed of any generation of all such transgenicinsect-resistant plants wherein said seed comprises a DNA sequenceencoding the polypeptide of the present invention is also an importantaspect of the invention.

The DNA constructs of the present invention may be introduced into thegenome of a desired plant host by a variety of conventionaltransformation techniques, which are well known to those skilled in theart. Preferred methods of transformation of plant cells or tissues arethe Agrobacterium mediated transformation method and the biolistics orparticle-gun mediated transformation method. Suitable planttransformation vectors for the purpose of Agrobacterium mediatedtransformation include those derived from a Ti plasmid of Agrobacteriumtumefaciens, as well as those disclosed, e.g., by Herrera-Estrella etal., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12: 8711-8721(1984); Klee et al., Bio-Technology 3(7): 637-642 (1985); and EPOpublication 120,516. In addition to plant transformation vectors derivedfrom the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternativemethods can be used to insert the DNA constructs of this invention intoplant cells. Such methods may involve, but are not limited to, forexample, the use of liposomes, electroporation, chemicals that increasefree DNA uptake, free DNA delivery via microprojectile bombardment, andtransformation using viruses or pollen.

A plasmid expression vector suitable for the introduction of a nucleicacid encoding a polypeptide or protein of the present invention inmonocots using electroporation or particle-gun mediated transformationis composed of the following: a promoter that is constitutive ortissue-specific; an intron that provides a splice site to facilitateexpression of the gene, such as the Hsp70 intron (PCT PublicationWO93/19189); and a 3′ polyadenylation sequence such as the nopalinesynthase 3′ sequence (NOS 3′; Fraley et al., Proc. Natl. Acad. Sci. USA80: 4803-4807 (1983)). This expression cassette may be assembled on highcopy replicons suitable for the production of large quantities of DNA.

An example of a useful Ti plasmid cassette vector for planttransformation is pMON17227. This vector is described in PCT PublicationWO 92/04449 and contains a gene encoding an enzyme conferring glyphosateresistance (denominated CP4), which is an excellent selection markergene for many plants. The gene is fused to the Arabidopsis EPSPSchloroplast transit peptide (CTP2) and expressed from the FMV promoteras described therein.

When adequate numbers of cells (or protoplasts) containing the exogenousnucleic acid encoding a polypeptide or protein of the present inventionare obtained, the cells (or protoplasts) are regenerated into wholeplants. Choice of methodology for the regeneration step is not critical,with suitable protocols being available for hosts from Leguminosae(alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery,parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice,maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), variousfloral crops, such as sunflower, and nut-bearing trees, such as almonds,cashews, walnuts, and pecans. See, for example, Ammirato et al.,Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. (1984);Shimamoto et al., Nature 338:274-276 (1989); Fromm, UCLA Symposium onMolecular Strategies for Crop Improvement, Apr. 16-22, 1990. Keystone,Colo. (1990); Vasil et al., Bio/Technology 8:429-434 (1990); Vasil etal., Bio/Technology 10:667-674 (1992); Hayashimoto, Plant Physiol.93:857-863 (1990); and Datta et al., Bio-technology 8:736-740 (1990).Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. Plant Phys. 38:467-486 (1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single exogenous gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedexogenous gene. More preferred is a transgenic plant that is homozygousfor the added exogenous gene; i.e., a transgenic plant that contains twoadded exogenous genes, one gene at the same locus on each chromosome ofa chromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selfing) an independent segregant transgenic plant thatcontains a single exogenous gene, germinating some of the seed producedand analyzing the resulting plants produced for the exogenous gene ofinterest.

The development or regeneration of transgenic plants containing theexogenous nucleic acid that encodes a polypeptide or protein of interestis well known in the art. Preferably, the regenerated plants areself-pollinated to provide homozygous transgenic plants, as discussedabove. Otherwise, pollen obtained from the regenerated plants is crossedto seed-grown plants of agronomically important lines. Conversely,pollen from plants of these important lines is used to pollinateregenerated plants. A transgenic plant of the present inventioncontaining a desired polypeptide or protein of the present invention iscultivated using methods well known to one skilled in the art.

Transgenic plants, that can be generated by practice of the presentinvention, include but are not limited to Acacia, alfalfa, aneth, apple,apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans,beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage,canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry,cilantro, citrus, clementines, coffee, corn, cotton, cucumber, Douglasfir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape,grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime,Loblolly pine, mango, melon, mushroom, nut, oat, okra, onion, orange, anornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet,sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco,tomato, turf, a vine, watermelon, wheat, yams, and zucchini.

The present invention also provides parts of the transgenic plants ofpresent invention. Plant parts, without limitation, include seed,endospeiin, ovule and pollen. In a particularly preferred embodiment ofthe present invention, the plant part is a seed.

The present invention also further provides method for generating atransgenic plant comprising the steps of: a) introducing into the genomeof the plant an exogenous nucleic acid, wherein the exogenous nucleicacid comprises in the 5′ to 3′ direction i) a promoter that functions inthe cells of said plant, said promoter operably linked to; ii) astructural nucleic acid sequence encoding a polypeptide or protein ofthe present invention, said structural nucleic acid sequence operablylinked to; iii) a 3′ non-translated nucleic acid sequence that functionsin said cells of said plant to cause transcriptional termination; b)obtaining transformed plant cells containing the nucleic acid sequenceof step (a); and c) regenerating from said transformed plant cells atransformed plant in which said polypeptide or protein is overexpressed.

Any of the isolated nucleic acid molecules of the present invention maybe introduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers etc. Further any of the nucleic acid molecules encoding aXenorhabdus protein or polypeptide of the present invention may beintroduced into a plant cell in a manner that allows for over expressionof the protein or polypeptide encoded by the nucleic acid molecule.

Antibodies have been expressed in plants (Hiatt et al., Nature 342:76-78(1989); Conrad and Fielder, Plant Mol. Biol. 26:1023-1030 (1994)).Cytoplasmic expression of a scFv (single-chain Fv antibodies) has beenreported to delay infection by artichoke mottled crinkle virus.Transgenic plants that express antibodies directed against endogenousproteins may exhibit a physiological effect (Philips et al., EMBO J.16:4489-4496 (1997); Marion-Poll, Trends in Plant Science 2:447-448(1997)). For example, expressed anti-abscisic antibodies reportedlyresult in a general perturbation of seed development (Philips et al.,EMBO J. 16:4489-4496 (1997)).

Antibodies that are catalytic may also be expressed in plants (abzymes).The principle behind abzymes is that since antibodies may be raisedagainst many molecules, this recognition ability can be directed towardgenerating antibodies that bind transition states to force a chemicalreaction forward (Persidas, Nature Biotechnology 15:1313-1315 (1997);Baca et al., Ann. Rev. Biophys. Biomol. Struct. 26:461-493 (1997)). Thecatalytic abilities of abzymes may be enhanced by site directedmutagenesis. Examples of abzymes are, for example, set forth in U.S.Pat. Nos. 5,658,753; 5,632,990; 5,631,137; 5,602,015; 5,559,538;5,576,174; 5,500,358; 5,318,897; 5,298,409; 5,258,289 and 5,194,585, allof which are herein incorporated in their entirety.

It is understood that any of the antibodies of the present invention maybe expressed in plants and that such expression can result in aphysiological effect. It is also understood that any of the expressedantibodies may be catalytic.

Microbial Constructs and Transformed Microbial Cells

The nucleotide sequences of the present invention may be introduced intoa wide variety of prokaryotic and eukaryotic microorganism hosts toexpress the Xenorhabdus or Photorhabdus polypeptide or protein ofinterest, particularly the insect inhibitory polypeptides or proteins ofthe present invention. The term “microorganism” includes prokaryotic andeukaryotic microbial species such as bacteria and fungi. Fungi includeyeast and filamentous fungi. Illustrative prokaryotes, bothGram-negative and Gram-positive, include Enterobacteriaceae, such asEscherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae;Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium,Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter;Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Amongeukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

For the purpose of plant protection against insects, a large number ofmicroorganisms known to inhabit the phylloplane (the surface of theplant leaves) and/or the rhizosphere (the soil surrounding plant roots)of a wide variety of important crops may also be desirable host cellsfor manipulation, propagation, storage, delivery and/or mutagenesis ofthe disclosed recombinant constructs. These microorganisms includebacteria, algae, and fungi. Of particular interest are microorganisms,such as bacteria, e.g., genera Bacillus (including the species andsubspecies B. thuringiensis kurstaki HD-1, B. thuringiensis kurstakiHD-73, B. thuringiensis sotto, B. thuringiensis berliner, B.thuringiensis thuringiensis, B. thuringiensis tolworthi, B.thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensisgalleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.thuringiensis entomocidus, B. thuringiensis tenebrionis and B.thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobactersphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans.

It is well known that exogenous nucleic acids encoding polypeptides ofinterest can be introduced into a microbial host cell, such as abacterial cell or a fungal cell, using a recombinant construct. Thepresent invention also relates to a fungal or bacterial recombinantconstruct comprising a structural nucleotide sequence encoding aXenorhabdus or Photorhabdus protein or polypeptide. In a preferredembodiment, the structural nucleotide sequence encodes an insectinhibitory protein or polypeptide of the present invention. The presentinvention also relates to a bacterial or fungal cell comprising abacterial or fungal recombinant vector. The present invention alsorelates to methods for obtaining a recombinant bacterial or fungal hostcell, comprising introducing into a bacterial or fungal host cell anexogenous nucleic acid molecule.

The bacterial recombinant vector may be a linear or a closed circularplasmid. The vector system may be a single vector or plasmid or two ormore vectors or plasmids which together contain the total DNA to beintroduced into the genome of the bacterial host. In addition, thebacterial vector may be an expression vector. Nucleic acid moleculesencoding Xenorhabdus proteins or polypeptide can, for example, besuitably inserted into a replicable vector for expression in a bacteriumunder the control of a suitable promoter for that bacterium. Manyvectors are available for this purpose, and selection of the appropriatevector will depend mainly on the size of the nucleic acid to be insertedinto the vector and the particular host cell to be transformed with thevector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the particularhost cell with which it is compatible. The vector components forbacterial transformation generally include, but are not limited to, oneor more of the following: a signal sequence, an origin of replication,one or more selectable marker genes, and an inducible promoter allowingthe expression of exogenous DNA.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with bacterial hosts. The vector ordinarily carries areplication site, as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (see, e.g., Bolivar et al., Gene 2:95 (1977)). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage, also generally contains,or is modified to contain, promoters that can be used by the microbialorganism for expression of the selectable marker genes.

Nucleic acid molecules encoding Xenorhabdus proteins or polypeptides maybe expressed not only directly, but also as a fusion with anotherpolypeptide, preferably a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature polypeptide. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the polypeptide encoding DNA that is inserted into thevector. The heterologous signal sequence selected should be one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For bacterial host cells that do not recognize and processthe native polypeptide signal sequence, the signal sequence issubstituted by a bacterial signal sequence selected, for example, fromthe group consisting of the alkaline phosphatase, β-lactamase, orheat-stable enterotoxin II leaders and the like.

Both expression and cloning vectors contain a nucleotide sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria.

Expression and cloning vectors also generally contain a selection gene,also termed a selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. One example of a selectionscheme utilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with a heterologous protein homologue orfragment thereof produce a protein conferring drug resistance and thussurvive the selection regimen.

The expression vector for producing a polypeptide can also contains aninducible promoter that is recognized by the host bacterial organism andis operably linked to the nucleic acid encoding, for example, thenucleic acid molecule encoding the Xenorhabdus protein or polypeptide ofinterest. Inducible promoters suitable for use with bacterial hostsinclude the β-lactamase, E. coli λ phage PL and PR, and E. coligalactose, arabinose, alkaline phosphatase, tryptophan (trp), andlactose operon promoter systems and variations thereof (Chang et al.,Nature 275:615 (1978); Goeddel et al., Nature 281:544 (1979); Guzman etal., J. Bacteriol. 174:7716-7728 (1992); Goeddel, Nucleic Acids Res.8:4057 (1980); EP 36,776) and hybrid promoters such as the tac promoter(deBoer et al., Proc. Natl. Acad. Sci. (USA) 80:21-25 (1983)). However,other known bacterial inducible promoters are suitable (Siebenlist etal., Ce1120:269 (1980)).

Promoters for use in bacterial systems also generally contain aShine-Dalgarno (S.D.) sequence or a consensus sequence thereof operablylinked to the DNA encoding the polypeptide of interest. The promoter canbe removed from the bacterial source DNA by restriction enzyme digestionand inserted into the vector containing the desired DNA coding sequence,or vice versa.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. For example, integrating vectors constructed withDNA from various Bacillus strains integrate into the Bacillus chromosome(E.P.O. Pub. No. 127,328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences.

Construction of suitable vectors containing one or more of theabove-listed components employs standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, axenorhabdus protein or polypeptide of the present invention, may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke and Schuster J. Biol. Chem.264:5503-5509 (1989)); and the like. pGEX vectors (Promega, MadisonWis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

It is, of course, necessary to select the appropriate bacteria takinginto consideration replicability of the replicon in the cells of abacterium. For example, E. coli, Serratia, or Salmonella species can besuitably used as the host when well known plasmids such as pBR322,pBR325, pACYC177, or pKN410 are used to supply the replicon. E. colistrain W3110 is a preferred host or parent host because it is a commonhost strain for recombinant DNA product fermentations. Preferably, thehost cell should secrete minimal amounts of proteolytic enzymes.

Host cells are transfected and preferably transformed with theabove-described vectors and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

Numerous methods of transfection are known to the ordinarily skilledartisan, for example, calcium phosphate and electroporation. Dependingon the host cell used, transformation is done using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in section 1.82 of Sambrook et al., MolecularCloning: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress, (1989), is generally used for bacterial cells that containsubstantial cell-wall barriers. Another method for transformationemploys polyethylene glycol/DMSO, as described in Chung and Miller(Chung and Miller, Nucleic Acids Res. 16:3580 (1988)). Yet anothermethod is the use of the technique termed electroporation. In addition,bacterial cells can be readily transformed using various forms of phages(i.e., transducing, temperate, lytic and lysogenic), suicide vectors forinserting DNA directly into the chromosome, and through homologousrecombination using either phages, suicide vectors or linear DNA.

Bacterial cells used to produce the polypeptide of interest for purposesof this invention are cultured in suitable media in which the promotersfor the nucleic acid encoding the heterologous polypeptide can beartificially induced as described generally, e.g., in Sambrook et al.,Molecular Cloning: A Laboratory Manual, New York: Cold Spring HarborLaboratory Press, (1989). Examples of suitable media are given in U.S.Pat. Nos. 5,304,472 and 5,342,763.

A yeast recombinant construct can typically include one or more of thefollowing: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence, a selectable marker. Theseelements can be combined into an expression cassette, which may bemaintained in a replicon, such as an extrachromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprocaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein et al., Gene,8:17-24 (1979)), pCl/1 (Brake et al., Proc. Natl. Acad. Sci. USA,81:4642-4646 (1984)), and YRp17 (Stinchcomb et al., J. Mol. Biol.,158:157 (1982)). In addition, a replicon may be either a high or lowcopy number plasmid. A high copy number plasmid will generally have acopy number ranging from about 5 to about 200, and typically about 10 toabout 150. A host containing a high copy number plasmid will preferablyhave at least about 10, and more preferably at least about 20.

Useful yeast promoter sequences can be derived from genes encodingenzymes in the metabolic pathway. Examples of such genes include alcoholdehydrogenase (ADH) (E.P.O. Pub. No. 284044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (E.P.O. Pub. No. 329203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promotersequences (Myanohara et al., Proc. Natl. Acad. Sci. USA, 80:1 (1983)).In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. Examples of such hybrid promoters includethe ADH regulatory sequence linked to the GAP transcription activationregion (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples ofhybrid promoters include promoters which consist of the regulatorysequences of either the ADH2, GAL4, GAL10, or PHO5 genes, combined withthe transcriptional activation region of a glycolytic enzyme gene suchas GAP or PyK (E.P.O. Pub. No. 164556). Furthermore, a yeast promotercan include naturally occurring promoters of non-yeast origin that havethe ability to bind yeast RNA polymerase and initiate transcription.Examples of such promoters include, inter alia, (Cohen et al., Proc.Natl. Acad. Sci. USA, 77:1078 (1980); Henikoff et al., Nature 283:835(1981); Hollenberg et al., Curr. Topics Microbiol. Immunol., 96:119(1981); Mercerau-Puigalon et al., Gene, 11:163 (1980); and Panthier etal., Curr. Genet., 2:109 (1980)).

Intracellularly expressed fusion proteins provide an alternative todirect expression of the polypeptides of interest. Typically, a DNAsequence encoding the N-terminal portion of a stable protein, a fusionpartner, is fused to the 5′ end of heterologous structural nucleotidesequence encoding the desired polypeptide. Upon expression, thisconstruct will provide a fusion of the two amino acid sequences. The DNAsequence at the junction of the two amino acid sequences may or may notencode a cleavable site. See, e.g., E.P.O. Pub. No. 196056. Anotherexample is a ubiquitin fusion protein. Such a ubiquitin fusion proteinpreferably retains a site for a processing enzyme (e.g.ubiquitin-specific processing protease) to cleave the ubiquitin from thepolypeptide of the present invention. Through this method, therefore, amature polypeptide can be isolated [see, P.C.T. WO 88/024066].

Alternatively, polypeptides or proteins can also be secreted from thecell into the growth media by creating chimeric DNA molecules thatencode a fusion protein comprised of a leader sequence fragment thatprovides for secretion in yeast of the polypeptides. Preferably, thereare processing sites encoded between the leader fragment and thepolypeptide-encoding sequence fragment that can be cleaved either invivo or in vitro. The leader sequence fragment typically encodes asignal peptide comprised of hydrophobic amino acids which direct thesecretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (E.P.O. Pub.No. 12873; J.P.O. Pub. No. 62,096,086) and the A-factor gene (U.S. Pat.No. 4,588,684). Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast(E.P.O. Pub. No. 60057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(typically about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,546,083 and 4,870,008; and E.P.O. Pub. No. 324274). Additional leadersemploying an alpha-factor leader fragment that provides for secretioninclude hybrid alpha-factor leaders made with a pre-sequence of a firstyeast, but a pro-region from a second yeast alpha factor. See, e.g.,P.C.T. WO 89/02463.

Examples of transcription terminator sequence and other yeast-recognizedtermination sequences, such as those coding for glycolytic enzymes, areknown to those of skill in the art.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome (Orr-Weaver et al., Methods in Enzymol., 101:228-245(1983)). An integrating vector may be directed to a specific locus inyeast by selecting the appropriate homologous sequence for inclusion inthe vector. See On-Weaver et al., supra. One or more expressionconstructs may integrate, possibly affecting levels of recombinantprotein produced (Rine et al., Proc. Natl. Acad. Sci. USA, 80:6750(1983)). The chromosomal sequences included in the vector can occureither as a single segment in the vector, which results in theintegration of the entire vector, or as two segments homologous toadjacent segments in the chromosome and flanking the expressionconstruct in the vector, which results in the stable integration of onlythe expression construct.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans (Kurtz, et al., Mol. Cell.Biol., 6:142 (1986)), Candida maltosa (Kunze et al., J. BasicMicrobiol., 25:141 (1985)); Hansenula polymorpha (Gleeson et al., J.Gen. Microbiol. 132:3459 (1986); Roggenkamp et al., Mol. Gen. Genet.202:302 (1986)); Kluyveromyces fragilis (Das et al., J. Bacteriol.158:1165 (1984)); Kluyveromyces lactis (De Louvencourt et al., J.Bacteriol. 154:737 (1983); Van den Berg et al., Bio/Technology 8:135(1990)); Pichia guillerimondii (Kunze et al., J. Basic Microbiol. 25:141(1985)); Pichia pastoris (Cregg et al., Mol. Cell. Biol. 5:3376 (1985);U.S. Pat. Nos. 4,837,148 and 4,929,555); Saccharomyces cerevisiae(Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978); Ito et al.,J. Bacteriol. 153:163 (1983)); Schizosaccharomyces pombe (Beach andNurse, Nature 300:706 (1981)); and Yarrowia lipolytica (Davidow, et al.,Curr. Genet. 10:380471 (1985); and Gaillardin et al., Curr. Genet. 10:49(1985)).

Methods of introducing exogenous nucleic acids into yeast hosts arewell-known in the art, and typically include either the transformationof spheroplasts or of intact yeast cells treated with alkali cations.Transformation procedures usually vary with the yeast species to betransformed. See e.g., Kurtz et al., Mol. Cell. Biol. 6:142 (1986);Kunze et al., J. Basic Microbiol. 25:141 (1985) for Candida. See, e.g.,Gleeson et al., J. Gen. Microbiol. 132:3459 (1986); Roggenkamp et al.,Mol. Gen. Genet. 202:302 (1986) for Hansenula. See, e.g., Das et al., J.Bacteriol. 158:1165 (1984); De Louvencourt et al., J. Bacteriol.154:1165 (1983); Van den Berg et al., Bio/Technology 8:135 (1990) forKluyveromyces. See, e.g., Cregg et al., Mol. Cell. Biol. 5:3376 (1985);Kunze et al., J. Basic Microbiol. 25:141 (1985); U.S. Pat. Nos.4,837,148 and 4,929,555 for Pichia. See, e.g., Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929 (1978); Ito et al., J. Bacteriol. 153:163(1983) for Saccharomyces. See, e.g., Beach and Nurse, Nature 300:706(1981) for Schizosaccharomyces. See, e.g., Davidow et al., Curr. Genet.10:39 (1985); Gaillardin et al., Curr. Genet. 10:49 (1985) for Yarrowia.

In order to obtain expression polypeptides or proteins of interest,recombinant microbial host cells derived from the transformants areincubated under conditions which allow expression of the recombinantpolypeptide-encoding sequence. These conditions will vary, dependentupon the host cell selected. However, the conditions are readilyascertainable to those of ordinary skill and knowledge in the art.

Detection of polypeptides expressed in the transformed host cell may beperformed by several methods. For example, a polypeptide or protein maybe detected by its immunological reactivity with antibodies.

Polypeptides or proteins of the present invention may be isolated fromthe cell by lysis, if formed intracellularly, or isolated from theculture medium, if secreted, by conventional methods.

Computer Media

The nucleotide sequence provided in SEQ ID NO:1 or fragment thereof, orcomplement thereof, or a nucleotide sequence at least 90% identical,preferably 95% or 98% identical, even more preferably 99% or 100%identical to the sequence provided in SEQ ID NO:1 or fragment thereof,or complement thereof, can be “provided” in a variety of mediums tofacilitate use. Such a medium can also provide a subset thereof in aform that allows a skilled artisan to examine the sequences.

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium that can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard disc,storage medium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of data processor structuring formats (e.g.text file or database) in order to obtain computer readable mediumhaving recorded thereon the nucleotide sequence information of thepresent invention.

By providing one or more of nucleotide sequences of the presentinvention, a skilled artisan can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.17:203-207 (1993)) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) within the genome that containhomology to ORFs or proteins from other organisms. Such ORFs areprotein-encoding fragments within the sequences of the present inventionand are useful in producing commercially important proteins such asenzymes used in amino acid biosynthesis, metabolism, transcription,translation, RNA processing, nucleic acid and a protein degradation,protein modification, and DNA replication, restriction, modification,recombination, and repair.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the nucleic acid molecule of the present invention. As usedherein, “a computer-based system” refers to the hardware means, softwaremeans, and data storage means used to analyze the nucleotide sequenceinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention comprises a centralprocessing unit (CPU), input means, output means, and data storagemeans. A skilled artisan can readily appreciate that any one of thecurrently available computer-based system are suitable for use in thepresent invention.

As indicated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein a nucleotidesequence of the present invention and the necessary hardware means andsoftware means for supporting and implementing a search means. As usedherein, “data storage means” refers to memory that can store nucleotidesequence information of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the nucleotidesequence information of the present invention. As used herein, “searchmeans” refers to one or more programs which are implemented on thecomputer-based system to compare a target sequence or target structuralmotif with the sequence information stored within the data storagemeans. Search means are used to identify fragments or regions of thesequence of the present invention that match a particular targetsequence or target motif A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are available can be used in the computer-based systems ofthe present invention. Examples of such software include, but are notlimited to, MacPattern (EMBL), BLASTIN and BLASTIX (NCBIA). One of theavailable algorithms or implementing software packages for conductinghomology searches can be adapted for use in the present computer-basedsystems.

The most preferred sequence length of a target sequence is from about 10to 100 amino acids or from about 30 to 300 nucleotide residues. However,it is well recognized that during searches for commercially importantfragments of the nucleic acid molecules of the present invention, suchas sequence fragments involved in gene expression and proteinprocessing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequences the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzymatic active sites andsignal sequences. Nucleic acid target motifs include, but are notlimited to, promoter sequences, cis elements, hairpin structures andinducible expression elements (protein binding sequences).

Thus, the present invention further provides an input means forreceiving a target sequence, a data storage means for storing the targetsequences of the present invention sequence identified using a searchmeans as described above, and an output means for outputting theidentified homologous sequences. A variety of structural formats for theinput and output means can be used to input and output information inthe computer-based systems of the present invention. A preferred formatfor an output means ranks fragments of the sequence of the presentinvention by varying degrees of homology to the target sequence ortarget motif. Such presentation provides a skilled artisan with aranking of sequences which contain various amounts of the targetsequence or target motif and identifies the degree of homology containedin the identified fragment.

A variety of comparing means can be used to compare a target sequence ortarget motif with the data storage means to identify sequence fragmentssequence of the present invention. For example, implementing softwarewhich implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol.Biol. 215:403-410 (1990)) can be used to identify open frames within thenucleic acid molecules of the present invention. A skilled artisan canreadily recognize that any one of the publicly available homology searchprograms can be used as the search means for the computer-based systemsof the present invention.

Exemplary Uses of the Agents of the Present Invention

Nucleic acid molecules and fragments thereof of the present inventionmay be employed to obtain other nucleic acid molecules from the same orclosely related species. Such nucleic acid molecules include the nucleicacid molecules that encode the complete coding sequence of a protein andpromoters and flanking sequences of such molecules. In addition, suchnucleic acid molecules include nucleic acid molecules that encode forother isozymes or gene family members. Such molecules can be readilyobtained by using the above-described nucleic acid molecules orfragments thereof to screen genomic libraries obtained from Xenorhabdus.Methods for forming such libraries are well known in the art.

Nucleic acid molecules and fragments thereof of the present inventionmay also be employed to obtain other nucleic acid molecules such asnucleic acid homologues. Such homologues include the nucleic acidhomologues of non-Xenorhabdus species including the nucleic acidmolecules that encode, in whole or in part, protein homologues of otherspecies or other organisms, sequences of genetic elements such aspromoters and transcriptional regulatory elements. Such molecules can bereadily obtained by using the above-described nucleic acid molecules orfragments thereof to screen cDNA or genomic libraries. Methods forforming such libraries are well known in the art. Such homologuemolecules may differ in their nucleotide sequences from those found inone or more of the nucleotide sequences disclosed in US PatentApplication Publication No. 20110167520 A1, which is incorporated hereinby reference in its entirety, or complements thereof because completecomplementarity is not needed for stable hybridization. The nucleic acidmolecules of the present invention therefore also include moleculesthat, although capable of specifically hybridizing with the nucleic acidmolecules may lack “complete complementarity.” In a particularembodiment, methods or 3′ or 5′ RACE may be used (Frohman, M. A. et al.,Proc. Natl. Acad. Sci. (U.S.A.) 85:8998-9002 (1988); Ohara, O. et al.,Proc. Natl. Acad. Sci. (U.S.A.) 86:5673-5677 (1989)) to obtain suchsequences.

Any of a variety of methods may be used to obtain one or more of theabove-described nucleic acid molecules (Zamechik et al., Proc. Natl.Acad. Sci. (U.S.A.) 83:4143-4146 (1986); Goodchild et al., Proc. Natl.Acad. Sci. (U.S.A.) 85: 5507-5511 (1988); Wickstrom et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:1028-1032 (1988); Holt et al., Molec. Cell. Biol.8:963-973 (1988); Gerwirtz et al., Science 242: 1303-1306 (1988);Anfossi et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:3379-3383 (1989);Becker et al., EMBO J. 8:3685-3691 (1989)). Automated nucleic acidsynthesizers may be employed for this purpose. In lieu of suchsynthesis, the disclosed nucleic acid molecules may be used to define apair of primers that can be used with the polymerase chain reaction(Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986);Erlich et al., European Patent 50,424; European Patent 84,796, EuropeanPatent 258,017; European Patent 237,362; Mullis, European Patent201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No.4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194) to amplify andobtain any desired nucleic acid molecule or fragment.

The nucleic acid molecules of the present invention may be used forphysical mapping. Physical mapping, in conjunction with linkageanalysis, can enable the isolation of genes. Physical mapping has beenreported to identify the markers closest in terms of geneticrecombination to a gene target for cloning. Once a DNA marker is linkedto a gene of interest, the chromosome walking technique can be used tofind the genes via overlapping clones. For chromosome walking, randommolecular markers or established molecular linkage maps are used toconduct a search to localize the gene adjacent to one or more markers. Achromosome walk (Bukanov and Berg, Mo. Microbiol. 11:509-523 (1994);Birkenbihl and Vielmetter Nucleic Acids Res. 17:5057-5069 (1989); Wenzeland Herrmann, Nucleic Acids Res. 16:8323-8336 (1988) is then initiatedfrom the closest linked marker. Starting from the selected clones,labeled probes specific for the ends of the insert DNA are synthesizedand used as probes in hybridizations against a representative library.Clones hybridizing with one of the probes are picked and serve astemplates for the synthesis of new probes; by subsequent analysis,contigs are produced.

The degree of overlap of the hybridizing clones used to produce a contigcan be determined by comparative restriction analysis. Comparativerestriction analysis can be carried out in different ways all of whichexploit the same principle; two clones of a library are very likely tooverlap if they contain a limited number of restriction sites for one ormore restriction endonucleases located at the same distance from eachother. The most frequently used procedures are, fingerprinting (Coulsonet al, Proc. Natl. Acad. Sci. (U.S.A.) 83:7821-7821, (1986); Knott etal., Nucleic Acids Res. 16:2601-2612 (1988); Eiglmeier et al., Mol.Microbiol. 7:197-206 (1993), 1993), restriction fragment mapping (Smithand Birnstiel, Nucleic Acids Res. 3:2387-2398 (1976)); or the“landmarking” technique (Charlebois et al. J. Mol. Biol. 222:509-524(1991)).

It is understood that the nucleic acid molecules of the presentinvention may in one embodiment be used in physical mapping. In apreferred embodiment, nucleic acid molecules of the present inventionmay in one embodiment be used in the physical mapping of Xenorhabdus.

Nucleic acid molecules of the present invention can be used incomparative mapping. Comparative mapping within families provides amethod to assess the degree of sequence conservation, gene order, ploidyof species, ancestral relationships and the rates at which individualgenomes are evolving. Comparative mapping has been carried out bycross-hybridizing molecular markers across species within a givenfamily. As in genetic mapping, molecular markers are needed but insteadof direct hybridization to mapping filters, the markers are used toselect large insert clones from a total genomic DNA library of a relatedspecies. The selected clones, each a representative of a single marker,can then be used to physically map the region in the target species. Theadvantage of this method for comparative mapping is that no mappingpopulation or linkage map of the target species is needed and the clonesmay also be used in other closely related species. By comparing theresults obtained by genetic mapping in model organisms, with those fromother species, similarities of genomic structure among species can beestablished. Cross-hybridization of RFLP markers has been reported andconserved gene order has been established in many studies. Suchmacroscopic synteny is utilized for the estimation of correspondence ofloci among these organisms. It is understood that markers of the presentinvention may in another embodiment be used in comparative mapping. In apreferred embodiment the markers of present invention may be used in thecomparative mapping of spore-forming Gram-positive bacteria.

In an aspect of the present invention, one or more of the agents of thepresent invention may be used to detecting the presence, absence orlevel of an organism, preferably a Xenorhabdus in a sample. In anotheraspect of the present invention, one or more of the nucleic molecules ofthe present invention are used to determine the level (i.e., theconcentration of mRNA in a sample, etc.) or pattern (i.e., the kineticsof expression, rate of decomposition, stability profile, etc.) of theexpression encoded in part or whole by one or more of the nucleic acidmolecule of the present invention (collectively, the “ExpressionResponse” of a cell or tissue). As used herein, the Expression Responsemanifested by a cell or tissue is said to be “altered” if it differsfrom the Expression Response of cells or tissues of organisms notexhibiting the phenotype. To determine whether a Expression Response isaltered, the Expression Response manifested by the cell or tissue of theorganism exhibiting the phenotype is compared with that of a similarcell or tissue sample of a organism not exhibiting the phenotype. Aswill be appreciated, it is not necessary to re-determine the ExpressionResponse of the cell or tissue sample of organisms not exhibiting thephenotype each time such a comparison is made; rather, the ExpressionResponse of a particular organism may be compared with previouslyobtained values of normal organism. As used herein, the phenotype of theorganism is any of one or more characteristics of an organism.

Nucleic acid molecules of the present invention can be used to monitorexpression. A microarray-based method for high-throughput monitoring ofgene expression may be utilized to measure gene-specific hybridizationtargets. This ‘chip’-based approach involves using microarrays ofnucleic acid molecules as gene-specific hybridization targets toquantitatively measure expression of the corresponding genes (Schena etal., Science 270:467-470 (1995); Shalon, Ph.D. Thesis, StanfordUniversity (1996)). Every nucleotide in a large sequence can be queriedat the same time. Hybridization can be used to efficiently analyzenucleotide sequences.

Several microarray methods have been described. One method compares thesequences to be analyzed by hybridization to a set of oligonucleotidesor cDNA molecules representing all possible subsequences (Bains andSmith, J. Theor. Biol. 135:303 (1989)). A second method hybridizes thesample to an array of oligonucleotide or cDNA probes. An arrayconsisting of oligonucleotides or cDNA molecules complementary tosubsequences of a target sequence can be used to determine the identityof a target sequence, measure its amount, and detect differences betweenthe target and a reference sequence. Nucleic acid molecules microarraysmay also be screened with protein molecules or fragments thereof todetermine nucleic acid molecules that specifically bind proteinmolecules or fragments thereof.

The microarray approach may also be used with polypeptide targets (U.S.Pat. No. 5,445,934; U.S. Pat. No. 5,143,854; U.S. Pat. No. 5,079,600;U.S. Pat. No. 4,923,901). Essentially, polypeptides are synthesized on asubstrate (microarray) and these polypeptides can be screened witheither protein molecules or fragments thereof or nucleic acid moleculesin order to screen for either protein molecules or fragments thereof ornucleic acid molecules that specifically bind the target polypeptides(Fodor et al., Science 251:767-773 (1991)).

It is understood that one or more of the molecules of the presentinvention, preferably one or more of the nucleic acid molecules orprotein molecules or fragments thereof of the present invention may beutilized in a microarray based method. In a preferred embodiment of thepresent invention, one or more of the Xenorhabdus nucleic acid moleculesor protein or polypeptide molecules or fragments thereof of the presentinvention may be utilized in a microarray based method. A particularpreferred microarray embodiment of the present invention is a microarraycomprising nucleic acid molecules encoding genes or fragments thereofthat are homologues of known genes or nucleic acid molecules thatcomprise genes or fragments thereof that elicit only limited or nomatches to known genes. A further preferred microarray embodiment of thepresent invention is a microarray comprising nucleic acid moleculeshaving genes or fragments thereof that are homologues of known genes andnucleic acid molecules that comprise genes or fragment thereof thatelicit only limited or no matches to known genes.

In a preferred embodiment, the microarray of the present inventioncomprises at least 10 nucleic acid molecules that specifically hybridizeunder stringent conditions to at least 10 nucleic acid moleculesencoding Xenorhabdus proteins or polypeptides or fragments thereof setforth in the tables of US Patent Application Publication No. 20110167520A1, which is incorporated herein by reference in its entirety. In a morepreferred embodiment, the microarray of the present invention comprisesat least 100 nucleic acid molecules that specifically hybridize understringent conditions to at least 100 nucleic acid molecules that encodea Xenorhabdus protein or polypeptide or fragment thereof set forththerein. In an even more preferred embodiment, the microarray of thepresent invention comprises at least 1,000 nucleic acid molecules thatspecifically hybridize under stringent conditions to at least 1,000nucleic acid molecules that encode a Xenorhabdus protein or polypeptideor fragment thereof set forth therein. In a further even more preferredembodiment, the microarray of the present invention comprises at least2,500 nucleic acid molecules that specifically hybridize under stringentconditions to at least 2,500 nucleic acid molecules that encode aXenorhabdus protein or polypeptide or fragment thereof set forththerein. While it is understood that a single nucleic acid molecule mayencode more than one protein homologue or fragment thereof, in apreferred embodiment, at least 50%, preferably at least 70%, morepreferably at least 80%, even more preferably at least 90% of thenucleic acid molecules that comprise the microarray contain one proteinor fragment thereof.

In a preferred embodiment, the microarray of the present inventioncomprises at least 10 nucleic acid molecules that specifically hybridizeunder stringent conditions to at least 10 nucleic acid moleculesselected from the nucleotide sequences disclosed in the aforementionedUS Patent Application Publication No. 20110167520 A1, or fragmentthereof or complement of either. In a more preferred embodiment, themicroarray of the present invention comprises at least 100 nucleic acidmolecules that specifically hybridize under stringent conditions to atleast 100 nucleic acid molecules that encode a Xenorhabdus protein orpolypeptide or fragment thereof set forth in the tables of theaforementioned US Patent Application Publication No. 20110167520 A1. Inan even more preferred embodiment, the microarray of the presentinvention comprises at least 1,000 nucleic acid molecules thatspecifically hybridize under stringent conditions to at least 1,000nucleic acid molecules that encode a Xenorhabdus protein or polypeptideor fragment thereof set forth therein. In a further even more preferredembodiment, the microarray of the present invention comprises at least2,500 nucleic acid molecules that specifically hybridize under stringentconditions to at least 2,500 nucleic acid molecules that encode aXenorhabdus protein or fragment thereof set forth therein. While it isunderstood that a single nucleic acid molecule may encode more than oneprotein homologue or fragment thereof, in a preferred embodiment, atleast 50%, preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90% of the nucleic acid molecules that comprisethe microarray contain one protein homologue or fragment thereof.

Nucleic acid molecules of the present invention may be used in sitedirected mutagenesis. Site-directed mutagenesis may be utilized tomodify nucleic acid sequences, particularly as it is a technique thatallows one or more of the amino acids encoded by a nucleic acid moleculeto be altered (e.g. a threonine to be replaced by a methionine). Threebasic methods for site-directed mutagenesis are often employed. Theseare cassette mutagenesis (Wells et al., Gene 34:315-23 (1985)); primerextension (Gilliam et al., Gene 12:129-137 (1980)); Zoller and Smith,Methods Enzymol. 100:468-500 (1983); and Dalbadie-McFarland et al.,Proc. Natl. Acad. Sci. (U.S.A.) 79:6409-6413 (1982)) and methods basedupon PCR (Scharf et al., Science 233:1076-1078 (1986); Higuchi et al.,Nucleic Acids Res. 16:7351-7367 (1988)). Site-directed mutagenesisapproaches are also described in European Patent 0 385 962, EuropeanPatent 0 359 472, and PCT Patent Application WO 93/07278.

Site-directed mutagenesis strategies have been applied to plants forboth in vitro as well as in vivo site-directed mutagenesis (Lanz et al.,J. Biol. Chem. 266:9971-9976 (1991); Kovgan and Zhdanov, Biotekhnologiya5: 148-154, No. 207160n, Chemical Abstracts 110: 225 (1989); Ge et al.,Proc. Natl. Acad. Sci. (U.S.A.) 86:4037-4041 (1989), Zhu et al., J.Biol. Chem. 271:18494-18498 (1996), Chu et al., Biochemistry33:6150-6157 (1994), Small et al., EMBO J. 11:1291-1296 (1992), Cho etal., Mol. Biotechnol. 8:13-16 (1997), Kita et al., J. Biol. Chem.271:26529-26535 (1996), Jin et al., Mol. Microbiol. 7:555-562 (1993),Hatfield and Vierstra, J. Biol. Chem. 267:14799-14803 (1992), Zhao etal., Biochemistry 31:5093-5099 (1992)).

Any of the nucleic acid molecules of the present invention may either bemodified by site-directed mutagenesis or used as, for example, nucleicacid molecules that are used to target other nucleic acid molecules formodification. It is understood that mutants with more than one alterednucleotide can be constructed using techniques that practitionersskilled in the art are familiar with such as isolating restrictionfragments and ligating such fragments into an expression vector (see,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989)). In a preferred embodiment of thepresent invention, one or more of the nucleic acid molecules orfragments thereof of the present invention may be modified bysite-directed mutagenesis.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor,N.Y.).

Insect inhibitory protein-encoding nucleic acids of the presentinvention will find particular uses in the plant protection againstinsects. For instance, insect-resistant transgenic plants can begenerated by introducing the exogenous nucleic acids encoding an insectinhibitory polypeptide or protein or insect inhibitory fragment thereof,the amino acid sequence of which is substantially identical to SEQ IDNO:2. Another example is to engineer transgenic microorganism (bacteriaor fungi) to express insect inhibitory polypeptides or proteins of thepresent invention and then to apply them to the insect food source orallow them to reside in soil surrounding plant roots or on the surfaceof plant leaves.

The transgenic microorganisms of the present invention may be used toproduce Xenorhabdus or Photorhabdus polypeptides or proteins ofinterest, particularly insect inhibitory polypeptides or proteins.Insect inhibitory polypeptides or proteins or insect inhibitoryfragments thereof may be secreted, for example as in bacterial systems,meaning targeted to either the periplasm as for gram negative bacteriaor localized to the extracellular space for gram negative or any othertype of bacterium, or localized to the intracellular spaces within thecytoplasm. Such compositions may be administered to insects according tomethods well known in the art. For example, insect inhibitorypolypeptides or proteins of the present invention may be formulated assprayable compositions or as a bait matrix.

The principle object of the present invention is to provide a method foridentification of any gene or any protein encoded by any structural genecontained within a Xenorhabdus or Photorhabdus species, particularlythose species which are shown to exhibit the production of an insectinhibitory protein or molecule or other similarly active composition,either alone or in combination with proteins or molecules or othersimilarly active compositions which may be derived from the bacterium inits role as natural symbiont within an insect pathogenic nematode host.

The present invention provides first for the isolation andidentification of one or more nematodes which have the capacity forinvading an insect larvae or adult, typically an insect of any orderincluding but not limited to Coleopteran, Dipteran, Hemipteran,Lepidopteran, Homopteran, Hymenopteran, or Lygus, white fly, or anysucking or piercing insect species. The isolation and identification ofa single insect pathogenic nematode species then enables the skilledartisan to isolate at least one species of Xenorhabdus or Photorhabdusendosymbiotic bacteria from the haemolymphe of an insect larvae or adultwhich has been invaded by the isolated and identified host nematode.

Nematodes can be isolated using methods particularly taught herein andin view of the prior art. Typically, one or more larvae of the genus andspecies Galleria melonella is/are placed into a soil sample under properhumidity and temperature conditions for a period of time to allow thelarvae to be invaded and colonized by a nematode species. It isgenerally believed from observations of nematode invasions that once asingle nematode species invades a single larvae or adult insect andsuccessfully releases bacterial symbionts of either Xenorhabdus orPhotorhabdus bacteria (which is further dependent upon the species ofhost nematode successfully invading the insect body), then an effectivecolonization of the insect haemolymphe by the bacterium so releasedresults in colonization of that particular insect host body to thatgenus and species of nematode and bacterium. This may be analogous tobacteriophage restriction inhibition, a phenomenon well known in thebacterial art, and may be a means for ensuring that the bacterium andnematode host-symbiont relationship is maintained.

The symbiont bacterium released into the haemolymphe then producesfungicides, acaricides, and antibacterial compounds which assist in thisrestriction of growth to the category and class or genus and species ofnematode and bacterium affiliated with the host-symbiont relationship.It may be that the nematode plays some functional role in thisrelationship, providing one or more specific factors, compositions, oraccessory proteins, small molecules or compositions which enable thereleased symbiont bacterium to sense the genus and species of the hostinsect body, possibly in association with factors, proteins, oraccessory compositions or receptors present within the haemolymphe ofthe pathogenized insect which enable the bacterium to switch on orexpress proteins which are effective in limiting the insect hosts'defenses, if any, which would otherwise lead to an ineffective invasionand infection by the nematode and bacterium. Particularly relevant tothis possibility is the presence as disclosed herein of several insectinhibitory proteins identified by their predicted structure, size andrelationship to previously identified Photorhabdus insect inhibitoryproteins from protein families such as Tcc, Tca, Tcb and Tcd (Ensign etal., WO 97/17432; and Ensign et al., WO 98/08932) encoded by an equalnumber of similar but substantially different genes. Interestingly, theinvasion of different insect genus/species hosts by a single insectpathogenic nematode which releases a single Xenorhabdus bacteriumspecies into the haemolymphe enables the skilled artisan to isolate thebacterium from each of the individual insect host bodies. Presumably,one skilled in classical microbiological methodologies would expect thatthe same bacterium now isolated and purified from each of the differentcadavers onto selective media, followed by the growth and analysis ofeach isolate in an identical broth medium for identical periods of timeand under identical conditions, would otherwise result in a similarprotein profile when analyzing the spent medium from such an experiment.However, the inventors herein have identified the surprising result thatthe protein profile of each isolate isolated and purified from differentcadaver species produce drastically different extracellular proteinprofiles when examined by 2D gel analysis. This result suggests thepossibility that the host insect, or the nematode, or the combination ofthe two, or alternatively merely the bacterium functions to sense itsenvironment and switches some genes on and others off to expressproteins and compositions which function in some particular yetunidentified manner specific for its host environment. The difference inprotein profiles of bacterium isolated from different cadaver species,yet presumably released as a single species from a single nematode hostmay be the result of at least a rearrangement the genome of thebacterium either by transposition or inversion or a combination of thetwo means to produce an otherwise isogenic line of bacterium capable ofexpressing an insect host selective combination of proteins which enablethe nematode-bacterium host-symbiont relationship to maximize itschances for survival. This result may also suggest that the nematode,although playing host to a bacterium which is capable of expressing avariety of proteins which individually are capable of inhibiting orkilling specific insect host species, may preferably invade a selectivegenus or species of insect for which it is more aptly suited in itsparticular host-symbiont relationship.

Nevertheless, the isolation and purification of an insect pathogenicnematode Xenorhabdus or Photorhabdus symbiont bacterium from an insectcadaver provides the basis for obtaining an amount of genomic DNA fromwhich a genomic library can be constructed to represent the entiregenome of the bacterial strain. The library can then be manipulated asdescribed herein to produce linear nucleotide sequences, which can thenbe compared to each other to identify regions of identity with which anoverlapping sequence can be generated to produce islands of linearsequence known as contigs because of the contiguous linear sequenceassembled from smaller bits of sequence data. The contigs can beassembled into a genomic map from which genes can be identified, andwherein translation of structural genes lead to further identificationof proteins having predicted structure and function based on homologiesof such predicted protein sequences as translated from open readingframes contained within the genome map, to proteins of known sequence,and perhaps also of known structure and function identified previouslyfrom other bacterial, viral, fungal, or other eukaryotic sources.

Syringomycins are bioactive lipodepsipeptides originally isolated fromthe phytopathogenic bacterium Pseudomonas syringae pv. Syringae. Thesecompounds are potent fungicides which inhibit the growth ofSaccharomyces cerevisiae and Aspergilus niger by forming pores in theplasma membrane. In addition, polypeptides generated from these proteinshave also been shown to have fungicidal activity. The Xenorhabdusgenomic DNA sequences disclosed herein contains at least 11 open readingframes encoding predicted proteins displaying homology to syringomycin.These open reading frames range in size from 4000-11, 500 base pairs andare exemplified by sequences shown in the tables in the US PatentApplication Publication No. 20110167520 A1, which is incorporated hereinby reference in its entirety. Any of these predicted proteins orpolypeptides derived from these proteins have potential antimicrobialactivity with commercial applications.

Insecticidal Compositions

The inventors contemplate that the Xenorhabdus strain and isolatableprotein compositions exhibiting insecticidal activity as disclosedherein will find particular utility as insecticides for topical and/orsystemic application to field crops, grasses, fruits and vegetables, andornamental plants. In a preferred embodiment, the bioinsecticidecomposition comprises an oil flowable suspension of bacterial cellswhich expresses a novel crystal protein disclosed herein. Preferably thecells are Xenorhabdus Xs85816 cells, however, any such bacterial hostcell expressing the novel nucleic acid segments disclosed herein andproducing an insecticidal protein is contemplated to be useful, such asB. thuringiensis, B. megaterium, B. subtilis, E. coli, Salmonellatyphimurium, other Xenorhabdus or Photorhabdus species, or Pseudomonasspp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel insecticidal protein disclosed herein.Preferred bacterial cells are Xenorhabdus Xs85816 cells, however,bacteria such as B. thuringiensis, B. megaterium, B. subtilis, E. coli,Salmonella typhimurium, other Xenorhabdus or Photorhabdus species, orPseudomonas spp. cells transformed with a DNA segment disclosed hereinand expressing the insecticidal protein are also contemplated to beuseful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novelinsecticidal protein disclosed herein. Preferred bacterial cells areXenorhabdus Xs85816 cells, however, bacteria such as B. thuringiensis,B. megaterium, B. subtilis, E. coli, Salmonella typhimurium, otherXenorhabdus or Photorhabdus species, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing theinsecticidal protein are also contemplated to be useful. Such dry formsof the insecticidal compositions may be formulated to dissolveimmediately upon wetting, or alternatively, dissolve in acontrolled-release, sustained-release, or other time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the insecticidal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the insecticidal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the Xenorhabdus gene. These cells may then beharvested in accordance with conventional ways. Alternatively, the cellscan be treated prior to harvesting.

When the insecticidal compositions comprise intact Xenorhabdus cellsexpressing the protein of interest, such bacteria may be formulated in avariety of ways. They may be employed as wettable powders, granules ordusts, by mixing with various inert materials, such as inorganicminerals (phyllosilicates, carbonates, sulfates, phosphates, and thelike) or botanical materials (powdered corncobs, rice hulls, walnutshells, and the like). The formulations may include spreader-stickeradjuvants, stabilizing agents, other pesticidal additives, orsurfactants. Liquid formulations may be aqueous-based or non-aqueous andemployed as foams, suspensions, emulsifiable concentrates, or the like.The ingredients may include rheological agents, surfactants,emulsifiers, dispersants, or polymers.

Alternatively, the novel Xip insecticidal proteins may be prepared bynative or recombinant bacterial expression systems in vitro and isolatedfor subsequent field application. Such protein may be either in crudecell lysates, suspensions, colloids, etc., or alternatively may bepurified, refined, buffered, and/or further processed, beforeformulating in an active biocidal formulation. Likewise, under certaincircumstances, it may be desirable to isolate insecticidal proteins orwhole cells from bacterial cultures expressing the insecticidal Xipprotein(s) and apply solutions, suspensions, or collodial preparationsof such insecticidal proteins or whole cells as the activebioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) is applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, or the specific piercing andsucking insect to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, insecticidal protein suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran or piercing and sucking insect,typically onto the foliage of the plant or crop to be protected, byconventional methods, preferably by spraying. The strength and durationof insecticidal application will be set with regard to conditionsspecific to the particular pest(s), crop(s) to be treated and particularenvironmental conditions. The proportional ratio of active ingredient tocarrier will naturally depend on the chemical nature, solubility, andstability of the insecticidal composition, as well as the particularformulation contemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 104 to about 107 cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1

Isolation of Entomopathogenic Nematodes

Entomopathogenic nematodes were isolated from soil samples obtained fromvarious geographic locations according to the following procedure.Generally the practice in the art is to infest soil samples with larvaeof the insect species Galleria mellonella. This example describes anentomopathogenic nematode baiting method not practiced in the art whichdoes not utilize Galleria mellonella, a non-pest and therefore anon-target insect species in baiting nematodes. Instead, this methoddescribes isolation of entomopathogenic nematodes having a greaterinsect inhibitory diversity by baiting with pest species and thereforetarget insect larvae. This method selects for nematode and Xenorhabdusand Photorhabdus bacterial strains having a greater diversity in theirinsect inhibitory properties and a greater diversity and variety ofinsect inhibitory proteins active against specific target insects.

Approximately 4 liters of soil sample by volume was placed into aplastic ziplock bag. The soil sample was then infested with a variety of4th instar target insects, e.g. corn ear worm, tobacco bud worm, blackcut worm, beet army worm, boll weevil, corn root worm, as well as thenon-target insect larvae Galleria mellonella. The insect larvae infestedsoil bags were zip sealed and incubated at 25° C. in the dark for about72 hours. Dead insect larvae were removed from the soil samples andidentified as to genus and species, washed in a mild NaOH/NaClO solution(50 ml water, 2 ml 4M NaOH, 1 ml 5% conc. NaClO), rinsed with sterilewater and placed into modified nematode traps (White, Science 66:302-303(1927)). The modified trap consists of the following elements assembledin order from bottom to top: (1) the bottom of a 4 inch diameter petridish half filled with water, (2) the lid of a 2 inch petri dish floatingin the water, (3) a dead insect larvae potentially infested withentomopathogenic nematodes placed onto a layer of Whatman filter paperlaying on the bottom of the inside of the smaller petri dish; (4)covered with the lid of the 4 inch diameter petri dish. This modified“White Trap” is incubated at 25° C. in the dark and left undisturbed for7-14 days. Infective juvenile stages of the nematodes begin to emergefrom the insect cadaver after one to two weeks and enter the watersurrounding the smaller diameter petri dish, forming a suspensioncontaining the nematodes. The nematode infested suspension is collected,placed into tissue culture flasks and these remain viable for severalmonths when stored at 12-16° C. in the dark.

As an example of the efficacy of this method, a Steinernema krausseinematode strain (#68) was previously isolated from a soil sample intowhich was placed only live Galleria mellonella larvae. A dead G.mellonella larvae was subsequently harvested and the sole nematodespecies isolated from the larvae was strain #68 (Mracek and Wester, J.Nematol. 25:710-717; 1993). Strain #68 was assayed for its insectinhibitory effects on larvae of the agricultural target insect pestscorn earworm, black cutworm and beet armyworm as well as Galleriamellonella. Six 4th instar larvae for each species were placedindividually into wells of a 24 well microtiter dish, each well beingbottom lined with a disc of Whatman filter paper. Ten microliters of astrain #68 nematode suspension, obtained and maintained as describedabove, was placed into each well and incubated in the dark at 25° C. forthree days. Larvae were then analyzed for survival, morbidity, andmortality. Unexpectedly, G. mellonella was not affected by nematodeinfestation, however all of the agricultural pest species were infectedand killed. The agricultural pest larvae were surface sterilized andtheir haemolymphe was streaked onto NBTA indicator agar plates. SingleXenorhabdus colonies were isolated and sub-cultured to eliminate thepresence of any possible contaminating bacteria and ensure pureXenorhabdus cultures. The Xenorhabdus bacteria isolated from cornearworm, black cutworm and beet armyworm were then grown for 48 hours inliquid BHI medium. In order to access both extracellular and cell boundcomponents which may be insect inhibitory, the culture was firstsubjected to a freeze thaw cycle, and then centrifuged and filtered (0.2μm), and the resulting lysate was separated from insoluble material. Thelysate was maintained at −70° C. unless used immediately. A 2D gelanalysis of the filtrate was completed to determine the protein profilesof each supernatant in triplicate. Surprisingly, bacterial proteinprofiles of bacteria harvested from corn earworm larvae were similar toeach other, but very different from the protein profiles of bacteriaisolated from either black cutworm or beet armyworm insect larvae. Thisresult suggests that a single strain of Xenorhabdus bacteria may beconditioned to express insect genus specific inhibitory proteins.

EXAMPLE 2

Isolation of Symbiotic Bacteria

Symbiotic bacteria were isolated from entomopathogenic nematodesaccording to the following procedure. A variety of 4th instar insectlarvae (corn ear worm, tobacco bud worm, black cut worm, beet army worm,boll weevil, corn root worm, and also Galleria mellonella) was placed ina 24 well plate containing Whatman filters in each well. Approx. 10 μlof entomopathogenic nematodes suspension was added into each wellcontaining one insect. The 24 well plates were sealed with parafilm andplaced at 25° C. in the dark.

After 48 to 72 hours dead insect larvae were removed from the 24-wellplate. The insect larvae were surface sterilized (20 ml water, 3 ml 4MNaOH and 1 ml 5% NaOCl) for 5 minutes and air-dried. The insect larvaewere cut open with sterile instruments on the lateral side withoutinjuring the gut and the haemolymphe was streaked on indicator agar(nutrient bromothymol blue agar and nutrient agar). The agar plates wereincubated at 30° C. in the dark for 48 hours.

Characteristic colonies were selected from the indicator plates: phase IXenorhabdus bacteria are able to take up bromothymol blue dye from thenutrient agar and form blue colonies. Bacterial characterization wasperformed according to methods known to the one skilled in the art(Farmer (1984), Bergey's Manual of Systematic Bacteriology, Vol. 1:510-511; Akhurst & Boemare (1988), J. Gen. Microbiol., Vol. 133:1835-1845; Boemare et al. (1993), Int. J. Syst. Bacteriol., Vol. 44:249-255).

Single characteristic phase I colonies were picked up by an inoculationloop and suspended into BHI media (Brain Heart Infusion medium (Difco),32 g/1, 50 ml in a 250 ml baffled flask). The bacteria were grown at 25°C. at 280 rpm on a rotary shaker in the dark. After 24 hours 15%glycerol was added to the bacterial culture, 1.5 ml aliquots for stockcultures were placed into cryovials and stored at −80° C.

EXAMPLE 3

Genomic Library Construction

Xenorhabdus strain Xs85816 was isolated and purified according tomethods described in examples 1 and 2 herein. Strain Xs85816 wasassociated with substantial insecticidal activity directed to lygus andboll weevil. Strain Xs85816 was deposited according to the BudapetsTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedures with the Agriculture ResearchCulture Collection (NRRL) International Depositary Authority at 1815North University Street, in Peoria, Ill. ZIP 61604 U.S.A. on Jun. 22,2000 and designated as NRRLB-30306, after having first been shown toexhibit insecticidal activity against piercing and sucking insects, inparticular against lygus species, and against boll weevil, and iscontemplated as a source for DNA sequences encoding insecticidalproteins, and when formulated into a composition of matter as a spray,powder or emulsion, for the treatment of plants or animals to inhibitinsect infestation. Xs85816 bacterial cells were grown in brain heartinfusion broth (Difco) for 42 hours at 25° C. to mid-exponential phase(OD650=˜1.0). Cells were poured into 10 1.5 ml-microfuge tubes and spunfor 5 minutes at ˜10,000 RPM to pellet. The supernatant was removed andthe cells were frozen. The frozen pellets were resuspended into 200 μlof TE (10 mM Tris 1 mM EDTA pH 8.0). Genomic DNA was prepared from thefrozen cell pellets using the Promega Genomic Preparation kit followingthe instructions of the manufacturer. Ten DNA samples were prepared fromthe cells above, and two of the samples were resuspended into 50 μl ofTE. Sample purity was tested and confirmed by digestion using therestriction enzymes EcoRI, HindIII, NotI, and SalI. The resuspendedsamples were used for the preparation of a genomic library.

The genomic library of Xenorhabdus strain Xs85816 was prepared accordingto standard procedures well known to those skilled in the art. ShearedDNA was polished with T4 polymerase and T4 polynucleotide kinase.Fragments 2-3 kb in length were recovered from an agarose gel. BstXIlinkers were then attached to the ends of the recovered fragments. BstXIlinkers consisted of two oligos capable of hybridizing to each otherover a portion of the length of one of the oligos, and providing a 3′four base overhang consisting of 5′-CACA-3′. Linker-ligated 2-3 kbfragments were gel purified and ligated into the BstXI digested plasmidvector pJCP2 and transformed into E. coli DH10B. BstXI cuts twice withinpJCP2, inactivating/removing a neomycin phosphotransferase codingsequence and leaving identical 3′ overhangs consisting of 5′-TGTG-3′.The resulting vector fragment contains an intact beta-lactamase codingsequence enabling selection of transformed cells containing genomicinsertions into the exposed BstXI overhangs on media containingampicillin Several ampicillin resistant transformants were selected andstreaked in duplicate onto media containing either ampicillin orkanamycin to determine the efficiency of the library construction.Greater than 95% of colonies arising from the transformation containedan insert, presumably derived from the genomic sequences. However, theinsertion frequency was probably somewhat lower because of theopportunity for the BstXI excised nptII coding sequence to re-insert inan inverted orientation or BstXI adapter itself to be ligated andinserted into plasmid. Approximately 200,000 colony forming units permicroliter of ligation mix were obtained. About thirty thousandindividual recombinant colonies were selected for DNA sequence analysisof inserted genomic DNA.

EXAMPLE 4

Generation and Assembly of Xenorhabdus sp. Genome Sequence

This example serves to illustrate the generation of the 1017 contigs andsingletons listed in the Sequence Listing. About 58000 genomicnucleotide sequence traces were derived from the double stranded plasmidlibrary as described in Example 3. The two basic methods for the DNAsequencing are the chain termination method of Sanger et al., Proc.Natl. Acad. Sci. (U.S.A.) 74:5463-5467 (1977) and the chemicaldegradation method of Maxam and Gilbert, Proc. Natl. Acad. Sci. (U.S.A.)74:560-564 (1977) using automated fluorescence-based sequencing asreported by Craxton, Method, 2:20-26 (1991); Ju et al., Proc. Natl.Acad. Sci. (U.S.A.) 92:4347-4351 (1995); and Tabor and Richardson, Proc.Natl. Acad. Sci. (U.S.A.) 92:6339-6343 (1995) and high speed capillarygel electrophoresis, e.g. as disclosed by Swerdlow and Gesteland,Nucleic Acids Res. 18:1415-1419 (1990); Smith, Nature 349:812-813(1991); Luckey et al., Methods Enzymol. 218:154-172 (1993); Lu et al.,J. Chromatog. A. 680:497-501 (1994); Carson et al., Anal. Chem.65:3219-3226 (1993); Huang et al., Anal. Chem. 64:2149-2154 (1992);Kheterpal et al., Electrophoresis 17:1852-1859 (1996); Quesada andZhang, Electrophoresis 17:1841-1851 (1996); Baba, Yakugaku Zasshi117:265-281 (1997). For instance, genomic nucleotide sequence traces aregenerated using a 377 or 3700 DNA Sequencer (Perkin-Elmer Corp., AppliedBiosystems Div., Foster City, Calif.) allowing for rapid electrophoresisand data collection. With these types of automated systems, fluorescentdye-labeled sequence reaction products are detected and chromatogramsare subsequently viewed, stored in a computer and analyzed usingcorresponding apparatus-related software programs. These methods areknown to those of skill in the art and have been described and reviewed(Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor,N.Y.

PHRED (phragment editor), which is developed by Phil Green at theUniversity of Washington, was used to call the bases from the sequencetrace files and to assign quality scores to the bases. PHRED usesFourier methods to examine the four base traces in the regionsurrounding each point in the data set in order to predict a series ofevenly spaced predicted locations. That is, it determines where thepeaks would be centered if there were no compressions, dropouts, orother factors shifting the peaks from their “true” locations. Next,PHRED examines each trace to find the centers of the actual, or observedpeaks and the areas of these peaks relative to their neighbors. Thepeaks are detected independently along each of the four traces so manypeaks overlap. A dynamic programming algorithm is used to match theobserved peaks detected in the second step with the predicted peaklocations found in the first step. Default parameters were used in thebase calling.

After the base calling is completed, sequence preprocessing is performedby removing 5′ and 3′ vector and linker sequences, according to standardprocedures well known in the art.

The preprocessed sequences were then assembled into contigs, or groupsof overlapping sequences. Contigs are assembled using PHRAP (phragmentassembly program) developed by Phil Green at the University ofWashington (http://www.mbt.washington.edu) using default assemblyparameters. This program takes a file of shotgun sequences and compilesconsensus contig sequences. Alignments are influenced by quality scores,based on Green's algorithm. Singletons are the remaining sequenceswithout sufficient overlaps with others after the assembly. The contigsand singletons files and their corresponding quality files were unitedto create “islands”.

A total of 1017 contigs and singletons were obtained. Contig sequencesare recognized as those sequences whose designations begin with XEN10C.Singleton sequences are recognized as those having designations whichbegin with gC-xewcLIB371. All contig and singleton sequences were runthrough the annotation and gene selection processes as described inExample 5.

EXAMPLE 5

Identification of Xenorhabdus sp. Genes

This example illustrates the identification of genes within the 1017contig and singleton sequences assembled as described in Example 4. Thegenes and partial genes embedded in such contigs and singletons wereidentified through a series of informatic analyses. Homology-basedsearches (i.e., BLASTX) were used to detect conserved sequences duringcomparisons of DNA sequences or hypothetically translated proteinsequences to public and/or proprietary DNA and protein databases.Existence of an Xenorhabdus sp. gene was inferred if significantsequence similarity extended over the majority of the target gene. Novelgenes, i.e., with no known homologs, were predicted with the programGeneMark, which calculates the probability of a gene based on thepresence of a gene-like ‘grammar’ in the DNA sequence (i.e., start andstop signals, and a significant open reading frame) and statisticalanalyses of protein-coding potential through biases in putative codonusage. The results of the homology and predictive methods were thenmerged into a single set of predicted coding regions, and their mostprobable translation.

The homology-based method used to define the Xenorhabdus sp. gene setwas BLASTX. For a description of BLASTX see Coulson, Trends inBiotechnology 12:76-80 (1994) and Birren et al., Genome Analysis,1:543-559 (1997). BLASTX takes a nucleotide sequence, translates it inthree forward reading frames and three reverse complement readingframes, and then compares the six translations against a proteinsequence database (e.g. the non-redundant protein (i.e., nr-aa) databasemaintained by the National Center for Biotechnology Information as partof GenBank and available at the web site: http://www.ncbi.nlm.nih.gov).BLASTX is run with the Xenorhabdus sp. contigs and singletons as queriesagainst the GenBank non-redundant protein data library identified as“nr-aa”. To identify genes solely by BLASTX, the maximum BLASTX E valueis set at 1E-08.

The ab initio method used to define the Xenorhabdus sp. gene set wasGeneMark. (see http://genemark.biology.gatech.edu/GeneMark for details).GeneMark uses inhomogeneous Markov chain models derived from comparisonsof known coding and non-coding sequences to predict the presence ofprotein-coding regions.

In Table 1 below (modified from Table 1 of the US Patent ApplicationPublication No. 20110167520 A1, which is incorporated herein byreference in its entirety), GroEL protein encoding regions in theXenorhabdus nucleic acid molecules of the present invention areidentified and results of the BLAST and GeneMark analyses provided.Where the predicted protein has a match to a homolog in thenon-redundant protein database, the confidence in accuracy of the geneprediction is proportional to the Bit score. “Bits” refers toinformation content, and the score in the “Bits” column indicates theamount of information in the hit. A higher bit score indicates a bettermatch. Low complexity matches (which can generate high BLAST scores ifthey match over long stretches with other low quality data) areinherently low information content, and hence do not generate high “bitscores”. Where the protein has been predicted by GeneMark, theconfidence in accuracy of the gene prediction is proportional to theGeneMark probability score. The higher the probability score, the morelikely the DNA sequence is transcribed into mRNA and translated intoprotein. Many, but not all, proteins are predicted by both BLASTX and byGeneMark. In these instances, both Bit scores and GeneMark probabilitiesare provided.

TABLE 1 Xenorhabdus Xs85816 Nucleic Acid Sequences, Encoded Proteins, &Annotations SEQ Contig/ Position: Polypepetide Prob. NCBI gi ID No.Singleton ID Start-Stop SEQ ID No. NCBI gi Bitscore E value %_ident%_cvrg (GM) Description 1 XEN10C258 653-1537 2 g6225121 476 1.00E−159 8951 0.87 60 KD CHAPERONIN (PROTEIN CPN60) (GROEL PROTEIN) [Enterobacteramnigenus] SEQ ID No.: Provides the SEQ ID NO listed in the SequenceListing for the contig or singleton in which the protein encoding regionis identified. Contig/Singleton ID: Provides the identification assignedto the contig or singleton in which the protein encoding region isidentified. Position: Start-Stop: Indicates location of the identifiedprotein encoding region within the contig or singleton. In cases wherethe first numeral is higher than its corresponding second numeral, theXenorhabdus sp. protein or fragment thereof is encoded by the complementof the sequence set forth in the sequence listing. Polypeptide SEQ IDNO: The sequence identification number of the amino acid sequence of thepolypeptide encoded by the identified open reading frame in the contigor singleton. NCBI gi: Refers to National Center for BiotechnologyInformation GenBank Identifier number which is the best match for agiven contig or singleton region from which the protein encoding regionwas identified. Bit Score: Bit score for BLAST match E value: Theexpected number of distinct segment pairs between two sequences with ascore above the bit score. %_ident: Refers to the percentage ofidentically matched nucleotides (or residues) that exist along thelength of that portion of the sequences which is aligned by the BLASTcomparison. %_cvrg: Refers to the percent of hit sequence length thatmatches to the query sequence in the match generated using BLAST (% cvrg= (match length/hit total length) × 100). Prob. (GM): A measure of thelikelihood that a region of DNA codes for a protein sequence asdetermined by the gene-predicting program GeneMark. NCBI gi Description:A description of the database entry referenced in the “NCBI gi” column.Sequences were analyzed by BLASTX against the non-redundant proteindatabase maintained by NCBI, and a description of the top hit isprovided.

The translation for each predicted protein into strings of amino acidsis provided. These predicted translations are the most probable, giventhe initiation and termination codons, and the biases in codon usageseen in publicly available Xenorhabdus genes.

Coding sequences identified in Table 1 of the aforementioned US PatentApplication Publication No. 20110167520 A1, encode many usefulXenorhabdus polypeptides or proteins, including but not limited toinsect inhibitory polypeptides or proteins, polypeptides or proteinscapable of conferring antibiotic resistance, cytotoxin proteins whichmay be used as microbial inhibitory proteins including bactericidal,bacteriostatic, fungicidal, and fungistatic polypeptides or proteins,polyketide synthases, polypeptides or proteins capable of conferringresistance to heavy metals or other toxic compositions, transposons andmobile genetic elements and their corresponding transposases, excisases,integrases, and invertases, phage and phage particle proteins,transcription regulatory proteins, translation regulatory proteins, andother useful proteins homologous to proteins derived from Xenorhabdus,Photorhabdus, Serratia, Yersinia, Salmonella, E. coli, and Erwinia sp.Specifically, the coding sequence as set forth in SEQ ID NO:1 encodes aGroEL protein.

EXAMPLE 6

Functional utility of insect inhibitory proteins produced by Xenorhabdus(or Photorhabdus) was tested using the following or a similar procedure.50 ml BHI medium in a 250 ml baffled flask was inoculated with 1.5 mlbacterial stock culture and grown at 25° C. and 280 rpm on a rotaryshaker in the dark. After 48 hours the culture was frozen at −80° C. forat least 24 hours. The culture broth was then thawed, centrifuged at2600×g for 30 minutes at 4° C. and decanted from the cell and debrispellet. The broth was then sterile-filtered (0.2 μm) and dialyzed. Theculture supernatant was used without an additional concentration stepfor bioassays to evaluate insect inhibitory, fungicidal and bactericidalproperties. Larvae were obtained using insect eggs obtained fromcommercial sources, hatched and reared using conventional insectarymethods.

Insect inhibitory activity was observed against western corn rootworm(WCR) and cotton boll weevil which are members of the insect orderColeoptera. The WCR is a member of the family Chrysomelidae. Othermembers of the Chrysomelid family include the Colorado potato beetle,the flea and leaf beetles. The cotton boll weevil is a member of thefamily Curculionidae which includes stored grains pests such as the riceand maize weevils and billbugs. Other Coleoptera include wireworms,seed-feeding bruchids, and grubs.

Insect inhibitory activity against western corn rootworm larvae wastested as follows. Xenorhabdus culture supernatant, control medium (BHI)or Tris buffer, pH 7.0, was applied to the surface (about 0.38 cm2) of amodified artificial diet (Bioserv™; diet product F9757) in 20 μlaliquots. The plates were allowed to air-dry in a drying chamber (16-20°C.; 40-50% RH) and the wells were infested with single non-diapausingneonate western corn rootworm (Coleoptera: Diabrotica virgiferavirgifera LeConte) hatched from surface disinfested eggs (Pleau, M.,1999. Master of Science Thesis., Nutritional physiology of Diabroticavirgifera. Saint Louis University). Plates were sealed, placed in ahumidified growth chamber and maintained at 27° C. for the appropriateperiod (5-7 days). Mortality and stunting (0-3) scores were thenassessed and statistically analyzed (SAS institute, 1989-1997. User'smanual for JMP version 3.2). Generally, 24 insects per treatment wereused in all studies. Control mortality was generally less than 10%.

Insect inhibitory activity against the cotton boll weevill (Coleoptera:Anthomonas grandis) was tested as follows. Xenorhabdus supernatant,control medium (BHI) or tris, pH 7.0, were applied in 20 μl aliquots tothe surface of 200 ul of artificial diet (Bioserv™ Co., Frenchtown,N.J.; diet product F9247) and allowed to air-dry. Boll weevil eggs werethen placed into the wells, the wells sealed and the plates held at 27°C., 60% relative humidity (RH) for 6 days. An activity score, based onconfounding of frass production, growth and mortality was then assessedand analyzed statistically (SAS institute, 1989-1997. User's manual forJMP version 3.2). Control mortality ranged between 0-25%.

The bacterial culture supernatant was also active against Lepidopteranlarvae, such as the cotton bollworm, corn earworm, beet armyworm, andblack cutworm, which are members of the Noctuidae family. Other Noctuidsinclude the armyworms of the genus Spodoptera, and the loopers such asthe cabbage looper. Activity was also observed against the European cornborer, a member of the Pyralidae family. Other Pyralids include thesouthwestern corn borer, the rice yellow stem borer, the pink stemborer, leaf rollers, and the Asiatic striped stem borer. Other typicalmembers of the order Lepidoptera are the codling moth, clothes moth,Indian meal moth, cabbage worm, bagworm, Eastern tent caterpillar, sodwebworm, and tobacco and tomato hornworms.

Insect inhibitory activity against Lepidopteran larvae was tested asfollows. Xenorhabdus culture supernatant, control medium (BHI) and Trisbuffer, pH 7.0, were applied directly to the surface (about 0.38 cm2) ofstandard artificial Lepidopteran diet (Southland Products Incorporated,Lake Village Ark.; diet product Lepidopteran multi-species diet) in 20μl aliquots. The diet plates were allowed to air-dry in a drying chamber(16-20° C.; 40-50% RH). The test wells were then infested with insecteggs, suspended in agar, of tobacco bud worm (Heliothis virescens), cornear worm (Helicoverpa zea) or black cut worm (Agrotis ipsylon). In thecase of European corn borer (Lepidoptera: Ostrinia nubilalis), neonateswere hand infested into the wells at one neonate per well. Followinginfestation, diet plates were sealed, placed in a humidity controlledgrowth chamber and maintained in the dark at 27° C. for the appropriateperiod of time. Mortality and stunting measurements were scored at day 5and statistically analyzed (SAS institute, 1989-1997. User's manual forJMP version 3.2). Generally 24 insects per treatment were used in allstudies. Control mortality generally ranged from 0-12.5%.

Insect inhibitory activity was also demonstrated against Lygus bug, amember of the order Hemiptera. Other members of the order include thestink bugs, seed bugs, chinch bugs, and stainers.

Insect inhibitory activity against Lygus bug (Hemiptera: Lygushesperus), was tested as follows. Feeding domes were made using adome-making machine manufactured by Analytical Research Systems,Gainesville Fla. Briefly, the system uses a vacuum to form domes fromParafilm™ sheeting using an aluminum block template shaped in the formof a 96-well microtiter-plate. To each such formed dome was added 40 ulof a 1:10 (v/v) dilution of test solution in diet. The dome-moldedParafilm™ is then heat sealed with a sheet of Mylar. The resultingParafilm dome sheet (96-wells) is placed onto a 96-well flat-bottomedmicrotiter plate containing one Lygus nymph each. The assay is typicallyscored after 4 days for mortality and stunting (0-3). Insect inhibitionresults from all tests were shown in Table 7 of the US PatentApplication Publication No. 20110167520 A1, which is incorporated hereinby reference in its entirety.

EXAMPLE 7

This example illustrates the alignment of insect inhibitory amino acidsequences identified from publicly available databases to sequencesencoded by genomic sequences disclosed herein. Also, thermalamplification primers are described based on conserved regionsidentified in the alignments which can be used to isolate DNA sequencesencoding insect inhibitory proteins from both Xenorhabdus andPhotorhabdus species. Surprisingly, primers designed to isolate insectinhibitory proteins based on regions of substantial homology betweenproteins from diverse species fail to produce amplification productsfrom strains which are believed to be phylogenetically more closelyrelated and which have been shown to produce insect inhibitory proteinsactive against the same target pest insect species. In this example,Xenorhabdus and Photorhabdus strains other than strain Xs85816 and W14were selected for thermal amplification and southern blot analysis basedon their having demonstrated activity against southern corn rootworm.

Translation of Xenorhabdus genomic data indicated several sequencesencoding proteins which exhibited homologies to previously identifiedPhotorhabdus insect inhibitory polypeptides available in publicdatabases such as GenBank (see, for example, Table 2 of theaforementioned US Patent Application Publication No. 20110167520 A1).Thermal amplification primers were designed to amplify DNA sequencesfrom within insect inhibitory coding sequences. Based on Xenorhabdussequences which were aligned with Photorhabdus sequences, the top BLASThits were used along with regions of greatest amino acid sequenceconservation for primer design. Xenorhabdus polypeptide XIP8 mostclosely aligned with polypeptide TccB from Photorhabdus strain W14.Based on this alignment, regions of greatest amino acid sequencehomology were used to design a XIP-8 primer set consisting of theprimers as disclosed in the US Patent Application Publication No.20110167520 A1, which is incorporated herein by reference in itsentirety. As indicated therein, this primer set amplified DNA xip8sequences from Xenorhabdus strain Xs85816 but not from other Xenorhabdusstrains or from at least one other Photorhabdus strain. Interestingly, aprimer set designed from regions of identity between XIP9 and XIP10protein coding sequences comprising the primers as disclosed in theaforementioned US Patent Application Publication No. 20110167520 A1 wasable to amplify xip9 and xip10 coding sequences from Xenorhabdus strainXs85816 as well as sequences from several other Xenorhabdus strains.However, the XIP9/10 primer set failed to amplify sequences from everyXenorhabdus or Photorhabdus strain analyzed, even though degeneracies(R=A+G, Y=C+T, W=A+T) were engineered into the primer set to compensatefor possible wobble. A further primer set similar in nature to the setdesigned for XIP9 and XIP10 was also designed from DNA sequencesencoding regions of amino acid sequence identity between XIP1 and XIP2consisting of the primers as disclosed in the aforementioned US PatentApplication Publication No. 20110167520 A1. Primer set XIP1/2 failed toamplify any sequences from Xenorhabdus or Photorhabdus strains otherthan Xenorhabdus strain Xs85816.

The product derived from thermal amplification of Xs85816 genomic DNAusing the primers designed to amplify XIP8 coding sequences was labeledand used to probe total genomic DNA from Xs85816 as well as otherXenorhabdus and Photorhabdus strains. A thermal amplification productderived from Xenorhabdus strain Xs85816 ompR, a highly conserved gene atthe DNA sequence level among gram negative Enterobacteriaceae, was usedas a control. The ompR sequence was able to hybridize to sequences inall strains analyzed, however, the xip8 sequence failed to hybridize toany sequences in the strains tested other than Xs85816. In addition,ompR thermal amplification primers based on the ompR gene in Xs85816also amplified a sequence of equivalent size from each strain tested.These results, taken together, suggest a large diversity in the proteinsexhibiting insect inhibitory activity from both Xenorhabdus andPhotorhabdus species.

What is claimed is:
 1. An isolated nucleic acid molecule comprising: a)a nucleic acid encoding a GroEL polypeptide having at least 95% identitywith the amino acid sequence of SEQ ID NO: 2, wherein said GroELpolypeptide has chaperonin activity; or b) the complement of the nucleicacid of a).
 2. The nucleic acid molecule of claim 1, wherein saidnucleic acid has at least 95% sequence identity with SEQ ID NO:
 1. 3.The nucleic acid molecule of claim 2, wherein said nucleic acid has atleast 98% sequence identity with SEQ ID NO:
 1. 4. The nucleic acidmolecule of claim 3, wherein said nucleic acid is 100% identical to SEQID NO:
 1. 5. A transformed host cell comprising the nucleic acidmolecule of claim
 1. 6. The transformed host cell of claim 5, whereinsaid host cell is selected from the group consisting of a plant cell, amammalian cell, a bacterial cell, an algal cell, an insect cell an afungal cell.
 7. A transgenic plant comprising the nucleic acid moleculeof claim
 1. 8. The transgenic plant of claim 7, further comprising a 5′non-coding sequence which functions in a plant to cause the productionof a mRNA molecule, said 5′ non-coding sequence operably linked to saidnucleic acid molecule, which is further operably linked to a 3′non-translated sequence that functions in said plant to causetermination of transcription.
 9. The transgenic plant of claim 7,wherein said plant is a monocot or a dicot plant.
 10. A seed producedfrom the plant of claim 7, wherein said seed comprises said nucleic acidmolecule.
 11. A progeny plant from the seed of claim 10, wherein saidprogeny comprises said nucleic acid molecule.